bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2022‒07‒10
fourteen papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute

  1. Cancer Res. 2022 Jul 06. pii: can.21.2908. [Epub ahead of print]
      Many advanced therapeutics possess cytostatic properties that suppress cancer cell growth without directly inducing death. Treatment-induced cytostatic cancer cells can persist and constitute a reservoir from which recurrent growth and resistant clones can develop. Current management approaches primarily comprise maintenance and monitoring because strategies for targeting non-proliferating cancer cells have been elusive. Here, we utilized targeted therapy paradigms and engineered cytostatic states to explore therapeutic opportunities for depleting treatment-mediated cytostatic cancer cells. Sustained oncogenic AKT signaling was common, while non-essential, in treatment-mediated cytostatic cancer cells harboring PI3K-pathway mutations, which are associated with cancer recurrence. Engineering oncogenic signals in quiescent mammary organotypic models showed that sustained, aberrant activation of AKT sensitized cytostatic epithelial cells to proteasome inhibition. Mechanistically, sustained AKT signaling altered cytostatic state homeostasis and promoted an oxidative and proteotoxic environment, which imposed an increased proteasome dependency for maintaining cell viability. Under cytostatic conditions, inhibition of the proteasome selectively induced apoptosis in the population with aberrant AKT activation compared to normal cells. Therapeutically exploiting this AKT-driven proteasome vulnerability was effective in depleting treatment-mediated cytostatic cancer cells independent of breast cancer subtype, epithelial origin, and cytostatic agent. Moreover, transient targeting during cytostatic treatment conditions was sufficient to reduce recurrent tumor growth in spheroid and mouse models. This work identified an AKT-driven proteasome-vulnerability that enables depletion of persistent cytostatic cancer cells harboring PTEN/PI3K pathway mutations, revealing a viable strategy for targeting non-proliferating persistent cancer cell populations before drug resistance emerges.
  2. Brain. 2022 Jul 04. pii: awac082. [Epub ahead of print]
      Epilepsy is one of the most frequent neurological diseases, with focal epilepsy accounting for the largest number of cases. The genetic alterations involved in focal epilepsy are far from being fully elucidated. Here, we show that defective lipid signalling caused by heterozygous ultra-rare variants in PIK3C2B, encoding for the class II phosphatidylinositol 3-kinase PI3K-C2β, underlie focal epilepsy in humans. We demonstrate that patients' variants act as loss-of-function alleles, leading to impaired synthesis of the rare signalling lipid phosphatidylinositol 3,4-bisphosphate, resulting in mTORC1 hyperactivation. In vivo, mutant Pik3c2b alleles caused dose-dependent neuronal hyperexcitability and increased seizure susceptibility, indicating haploinsufficiency as a key driver of disease. Moreover, acute mTORC1 inhibition in mutant mice prevented experimentally induced seizures, providing a potential therapeutic option for a selective group of patients with focal epilepsy. Our findings reveal an unexpected role for class II PI3K-mediated lipid signalling in regulating mTORC1-dependent neuronal excitability in mice and humans.
    Keywords:  PI3K-C2B; class II PI3K; epilepsy; mTOR; variants
  3. Elife. 2022 Jul 05. pii: e74510. [Epub ahead of print]11
      Background: Lymphatic malformations (LMs) often pose treatment challenges due to a large size or a critical location that could lead to disfigurement, and there are no standardized treatment approaches for either refractory or unresectable cases.Methods: We examined the genomic landscape of a patient cohort of LMs (n = 30 cases) that underwent comprehensive genomic profiling using a large-panel next-generation sequencing assay. Immunohistochemical analyses were completed in parallel.
    Results: These LMs had low mutational burden with hotspot PIK3CA mutations (n = 20) and NRAS (n = 5) mutations being most frequent, and mutually exclusive. All LM cases with Kaposi sarcoma-like (kaposiform) histology had NRAS mutations. One index patient presented with subacute abdominal pain and was diagnosed with a large retroperitoneal LM harboring a somatic PIK3CA gain-of-function mutation (H1047R). The patient achieved a rapid and durable radiologic complete response, as defined in RECIST1.1, to the PI3Kα inhibitor alpelisib within the context of a personalized N-of-1 clinical trial (NCT03941782). In translational correlative studies, canonical PI3Kα pathway activation was confirmed by immunohistochemistry and human LM-derived lymphatic endothelial cells carrying an allele with an activating mutation at the same locus were sensitive to alpelisib treatment in vitro, which was demonstrated by a concentration-dependent drop in measurable impedance, an assessment of cell status.
    Conclusions: Our findings establish that LM patients with conventional or kaposiform histology have distinct, yet targetable, driver mutations.
    Funding: R.P. and W.A. are supported by awards from the Levy-Longenbaugh Fund. S.G. is supported by awards from the Hugs for Brady Foundation. This work has been funded in part by the NCI Cancer Center Support Grants (CCSG; P30) to the University of Arizona Cancer Center (CA023074), the University of New Mexico Comprehensive Cancer Center (CA118100), and the Rutgers Cancer Institute of New Jersey (CA072720). B.K.M. was supported by National Science Foundation via Graduate Research Fellowship DGE-1143953.
    Clinical trial number: NCT03941782.
    Keywords:  NGS; PI3Kα; genomics; human; lymphatic malformations; medicine
  4. Cell Commun Signal. 2022 Jul 07. 20(1): 102
      BACKGROUND: Circular dorsal ruffles (CDRs) are rounded membrane ruffles induced on the dorsal surfaces of cells stimulated by growth factors (GF). They can serve as signal platforms to activate AKT protein kinase. After GF stimulation, phosphatidylinositol 3-kinase (PI3K) generates phosphatidylinositol (3,4,5)-triphosphate (PIP3) in the plasma membrane. PIP3 accumulates inside CDRs, recruits AKT into the structures, and phosphorylates them (pAKT). Given the importance of the PI3K-AKT pathway in GF signaling, CDRs are likely involved in cell growth. Interestingly, some cancer cell lines express CDRs. We hypothesized that CDRs contribute to carcinogenesis by modulating the AKT pathway. In the present study, we identified CDR-expressing cancer cell lines and investigated their cellular functions.METHODS: CDR formation was examined in six cancer cell lines in response to epidermal growth factor (EGF) and insulin. The morphology of the CDRs was characterized, and the related signaling molecules were observed using confocal and scanning electron microscopy. The role of CDRs in the AKT pathway was studied using biochemical analysis. The actin inhibitor cytochalasin D (Cyto D) and the PI3K inhibitor TGX221 were used to block CDRs.
    RESULTS: GF treatment induced CDRs in the hepatocellular carcinoma (HCC) Hep3B cell line, but not in others, including HCC cell lines HepG2 and Huh7, and the LO2 hepatocyte cell line. Confocal microscopy and western blot analysis showed that the PI3K-PIP3-AKT pathway was activated at the CDRs and that receptor proteins were recruited to the structures. Cyto D and TGX221 completely blocked CDRs and partially attenuated GF-induced pAKT. These results indicate that CDRs regulate the receptor-mediated PI3K-AKT pathway in Hep3B cells and the existence of CDR-independent pAKT mechanisms.
    CONCLUSIONS: Our results showed that CDRs modulate the AKT pathway in Hep3B cells. Since CDRs were not observed in other HCC and hepatocyte cell lines, we propose that CDRs in Hep3B would determine the carcinoma characteristic of the cell by aberrantly triggering the AKT pathway. Signaling molecules involved in CDR formation are promising therapeutic targets for some types of HCC. Video abstract.
    Keywords:  AKT; Circular dorsal ruffles; Hep3B; PI3K
  5. Front Immunol. 2022 ;13 890073
      Background: Activated phosphoinositide 3 kinase (PI3K) -delta syndrome (APDS) is an inborn error of immunity with variable clinical phenotype of immunodeficiency and immune dysregulation and caused by gain-of-function mutations in PIK3CD. The hallmark of immune phenotype is increased proportions of transitional B cells and plasmablasts (PB), progressive B cell loss, and elevated levels of serum IgM.Objective: To explore unique B cell subsets and the pathomechanisms driving B cell dysregulation beyond the transitional B cell stage in APDS.
    Methods: Clinical and immunological data was collected from 24 patients with APDS. In five cases, we performed an in-depth analysis of B cell phenotypes and cultured purified naïve B cells to evaluate their survival, activation, Ig gene class switch recombination (CSR), PB differentiation and antibody secretion. We also analyzed PB differentiation capacity of sorted CD27-IgD- double-negative B (DNB) cells.
    Results: The patients had increased B cell sizes and higher proportions of IgM+ DNB cells than healthy controls (HC). Their naïve B cells exhibited increased death, impaired CSR but relatively normal PB differentiation. Upon stimulation, patient's DNB cells secreted a similar level of IgG but a higher level of IgM than DNB cells from HC. Targeted therapy of PI3K inhibition partially restored B cell phenotypes.
    Conclusions: The present study suggests additional mechanistic insight into B cell pathology of APDS: (1) decreased peripheral B cell numbers may be due to the increased death of naïve B cells; (2) larger B cell sizes and expanded DNB population suggest enhanced activation and differentiation of naïve B cells into DNB cells; (3) the impaired CSR yet normal PB differentiation can predominantly generate IgM-secreting cells, resulting in elevated IgM levels.
    Keywords:  B cell survival and activation; CD27-IgD- double-negative B cells; Ig gene class switch recombination; activated PI3Kδ syndrome (APDS); plasma cell differentiation
  6. Cell Rep. 2022 Jul 05. pii: S2211-1247(22)00826-9. [Epub ahead of print]40(1): 111032
      How mechanistic target of rapamycin complex 1 (mTORC1), a key regulator of cellular metabolism, affects dendritic cell (DC) metabolism and T cell-priming capacity has primarily been investigated in vitro, but how mTORC1 regulates this in vivo remains poorly defined. Here, using mice deficient for mTORC1 component raptor in DCs, we find that loss of mTORC1 negatively affects glycolytic and fatty acid metabolism and maturation of conventional DCs, particularly cDC1s. Nonetheless, antigen-specific CD8+ T cell responses to infection are not compromised and are even enhanced following skin immunization. This is associated with increased activation of Langerhans cells and a subpopulation of EpCAM-expressing cDC1s, of which the latter show an increased physical interaction with CD8+ T cells in situ. Together, this work reveals that mTORC1 limits CD8+ T cell priming in vivo by differentially orchestrating the metabolism and immunogenicity of distinct antigen-presenting cell subsets, which may have implications for clinical use of mTOR inhibitors.
    Keywords:  CD8(+) T cells; CP: Immunology; IL-12; Langerhans cells; MHCI; immunization; mTORC1; metabolism; type 1 conventional dendritic cells
  7. Mol Brain. 2022 Jul 04. 15(1): 59
      Phosphatidylinositol 3,4,5-trisphosphate (PIP3) is a lipid second messenger that is crucial for the synaptic plasticity underlying learning and memory in pyramidal neurons in the brain. Our previous study uncovered PIP3 enrichment in the dendritic spines of hippocampal pyramidal neurons in the static state using a fluorescence lifetime-based PIP3 probe. However, the extent to which PIP3 enrichment is preserved in different states has not been fully investigated. Here, we revealed that PIP3 accumulation in dendritic spines is strictly controlled even in an active state in which PIP3 is increased by glutamate stimulation and high potassium-induced membrane depolarization. Time-course PIP3 analysis clarified the gradual PIP3 accumulation in dendritic spines over days during neuronal development. Collectively, these results deepen our understanding of PIP3 dynamics in dendritic spines, and the dysregulation of the PIP3 gradient between dendritic spines and shafts could cause neuronal diseases and mental disorders, such as autism spectrum disorder.
    Keywords:  Dendritic spine; Fluorescence lifetime; Fluorescence resonance energy transfer; Hippocampus; Phosphatidylinositol 3,4,5-trisphosphate; Two-photon microscopy
  8. Autophagy. 2022 Jul 04.
      Although attenuated IGF1R (insulin-like growth factor 1 receptor) signaling has long been viewed to promote longevity in model organisms, adverse effects on the heart have been the subject of major concern. We observed that IGF1R is overexpressed in cardiac tissues from patients with end-stage non-ischemic heart failure, coupled to the activation of the IGF1R downstream effector AKT/protein kinase B and inhibition of ULK1 (unc-51 like autophagy activating kinase 1). Transgenic overexpression of human IGF1R in cardiomyocytes from mice initially induces physiological cardiac hypertrophy and superior function, but later in life confers a negative impact on cardiac health, causing macroautophagy/autophagy inhibition as well as impaired oxidative phosphorylation, thus reducing life expectancy. Treatment with the autophagy inducer and caloric restriction mimetic spermidine ameliorates most of these IGF1R-induced cardiotoxic effects in vivo. Moreover, inhibition of IGF1R signaling by means of a dominant-negative phosphoinositide 3-kinase (PI3K) mutant induces cardioprotective autophagy, restores myocardial bioenergetics and improves late-life survival. Hence, our results demonstrate that IGF1R exerts a dual biphasic impact on cardiac health, and that autophagy mediates the late-life geroprotective effects of IGF1R inhibition in the heart.
    Keywords:  Heart failure; IGF1R; PI3K; human; insulin signaling; longevity; mitochondrial dysfunction; mouse
  9. Nat Commun. 2022 Jul 08. 13(1): 3955
      Protein arginine methyltransferase 5 (PRMT5) is the primary methyltransferase generating symmetric-dimethyl-arginine marks on histone and non-histone proteins. PRMT5 dysregulation is implicated in multiple oncogenic processes. Here, we report that PRMT5-mediated methylation of protein kinase B (AKT) is required for its subsequent phosphorylation at Thr308 and Ser473. Moreover, pharmacologic or genetic inhibition of PRMT5 abolishes AKT1 arginine 15 methylation, thereby preventing AKT1 translocation to the plasma membrane and subsequent recruitment of its upstream activating kinases PDK1 and mTOR2. We show that PRMT5/AKT signaling controls the expression of the epithelial-mesenchymal-transition transcription factors ZEB1, SNAIL, and TWIST1. PRMT5 inhibition significantly attenuates primary tumor growth and broadly blocks metastasis in multiple organs in xenograft tumor models of high-risk neuroblastoma. Collectively, our results suggest that PRMT5 inhibition augments anti-AKT or other downstream targeted therapeutics in high-risk metastatic cancers.
  10. Allergol Immunopathol (Madr). 2022 ;50(4): 1-9
      Monoallelic loss-of-function (LOF) mutations in the phosphatidylinositol 3-kinase (PIK3R1) gene affecting the inter-Src homology 2 domain of the p85α regulatory subunit of phosphoinositide--3-kinase δ (PI3Kδ) cause the activated PI3K δ syndrome (APDS2). APDS2 is defined as a primary antibody deficiency, developmental abnormalities within the B and T lymph cell compartments, and immune dysregulation. The genetic defect of APDS2 is shared with that of the SHORT syndrome, characterized by short stature, joint hyperextensibility, ocular depression, Rieger anomaly, and delayed tooth eruption. LOF variants in an intronic splice site (c.1425+1G.C/A/T) in the PI3KR1 gene have been identified in patients affected with both APDS2 and SHORT syndrome. Herein, we report a novel c.1644-1648del (p.Asp548Glufs*6) variant in a pediatric patient with the APDS2-related immunodeficiency, who presents with mild phenotypic features of the SHORT syndrome, congenital chest wall deformity, and IgE-mediated food allergy. The same variant was also identified in the patient's hitherto asymptomatic mother, implicating an incomplete penetrance. Regular monitoring by a multidisciplinary team under the pediatric clinical immunologist's supervision to implement appropriate diagnostic procedures and treatment modalities is of paramount importance. Further studies are required to better define the genotype-phenotype correlation in patients with the PIK3R1 gene mutations and to better delineate the mutual relationship between APDS2 and the SHORT syndrome.
    Keywords:  APDS2; PIK3R1; SHORT syndrome; immune dysregulation; immunodeficiency
  11. Cell Stem Cell. 2022 Jul 07. pii: S1934-5909(22)00252-1. [Epub ahead of print]29(7): 1018-1030
      The mammalian embryo exhibits a remarkable plasticity that allows it to correct for the presence of aberrant cells, adjust its growth so that its size is in accordance with its developmental stage, or integrate cells of another species to form fully functional organs. Here, we will discuss the contribution that cell competition, a quality control that eliminates viable cells that are less fit than their neighbors, makes to this plasticity. We will do this by reviewing the roles that cell competition plays in the early mammalian embryo and how they contribute to ensure normal development of the embryo.
  12. Cell Rep. 2022 Jul 05. pii: S2211-1247(22)00842-7. [Epub ahead of print]40(1): 111048
      Tuberous sclerosis complex (TSC) is a multisystem tumor-forming disorder caused by loss of TSC1 or TSC2. Renal manifestations predominately include cysts and angiomyolipomas. Despite a well-described monogenic etiology, the cellular pathogenesis remains elusive. We report a genetically engineered human renal organoid model that recapitulates pleiotropic features of TSC kidney disease in vitro and upon orthotopic xenotransplantation. Time course single-cell RNA sequencing demonstrates that loss of TSC1 or TSC2 affects multiple developmental processes in the renal epithelial, stromal, and glial compartments. First, TSC1 or TSC2 ablation induces transitional upregulation of stromal-associated genes. Second, epithelial cells in the TSC1-/- and TSC2-/- organoids exhibit a rapamycin-insensitive epithelial-to-mesenchymal transition. Third, a melanocytic population forms exclusively in TSC1-/- and TSC2-/- organoids, branching from MITF+ Schwann cell precursors. Together, these results illustrate the pleiotropic developmental consequences of biallelic inactivation of TSC1 or TSC2 and offer insight into TSC kidney lesion pathogenesis.
    Keywords:  CP: Developmental biology; CRISPR-Cas9; Schwann cell precursors; TSC1; TSC2; angiomyolipomas; disease pathogenesis; renal cysts; renal organoids; scRNA-seq; tuberous sclerosis complex
  13. EMBO Rep. 2022 Jul 08. e54677
      The proliferation and differentiation of antigen-specific B cells, including the generation of germinal centers (GC), are prerequisites for long-lasting, antibody-mediated immune protection. Affinity for antigen determines B cell recruitment, proliferation, differentiation, and competitiveness in the response, largely through determining access to T cell help. However, how T cell-derived signals contribute to these outcomes is incompletely understood. Here, we report how the signature cytokine of follicular helper T cells, IL-21, acts as a key regulator of the initial B cell response by accelerating cell cycle progression and the rate of cycle entry, increasing their contribution to the ensuing GC. This effect occurs over a wide range of initial B cell receptor affinities and correlates with elevated AKT and S6 phosphorylation. Moreover, the resultant increased proliferation can explain the IL-21-mediated promotion of plasma cell differentiation. Collectively, our data establish that IL-21 acts from the outset of a T cell-dependent immune response to increase cell cycle progression and fuel cyclic re-entry of B cells, thereby regulating the initial GC size and early plasma cell output.
    Keywords:  B cells; IL-21; cell cycle; germinal center; plasma cells
  14. Nat Commun. 2022 Jul 07. 13(1): 3908
      Plasmids are one of the most commonly used platforms for genetic engineering and recombinant gene expression in bacteria. The range of available copy numbers for cloning vectors is largely restricted to the handful of Origins of Replication (ORIs) that have been isolated from plasmids found in nature. Here, we introduce two systems that allow for the continuous, finely-tuned control of plasmid copy number between 1 and 800 copies per cell: a plasmid with an anhydrotetracycline-controlled copy number, and a parallelized assay that is used to generate a continuous spectrum of 1194 ColE1-based copy number variants. Using these systems, we investigate the effects of plasmid copy number on cellular growth rates, gene expression, biosynthesis, and genetic circuit performance. We perform single-cell timelapse measurements to characterize plasmid loss, runaway plasmid replication, and quantify the impact of plasmid copy number on the variability of gene expression. Using our assay, we find that each plasmid imposes a 0.063% linear metabolic burden on their hosts, hinting at a simple relationship between metabolic burdens and plasmid DNA synthesis. Our systems enable the precise control of gene expression, and our results highlight the importance of tuning plasmid copy number as a powerful tool for the optimization of synthetic biological systems.