bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2022‒11‒13
29 papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute

  1. Proc Natl Acad Sci U S A. 2022 11 16. 119(46): e2215621119
      Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that perform multiple and important cellular functions. The protein investigated here belongs to class IA of the PI3Ks; it is a dimer consisting of a catalytic subunit, p110α, and a regulatory subunit, p85α, and is referred to as PI3Kα. The catalytic subunit p110α is frequently mutated in cancer. The mutations induce a gain of function and constitute a driving force in cancer development. About 80% of these mutations lead to single-amino-acid substitutions in one of three sites of p110α: two in the helical domain of the protein (E542K and E545K) and one at the C-terminus of the kinase domain (H1047R). Here, we report the cryo-electron microscopy structures of these mutants in complex with the p110α-specific inhibitor BYL-719. The H1047R mutant rotates its sidechain to a new position and weakens the kα11 activation loop interaction, thereby reducing the inhibitory effect of p85α on p110α. E542K and E545K completely abolish the tight interaction between the helical domain of p110α and the N-terminal SH2 domain of p85α and lead to the disruption of all p85α binding and a dramatic increase in flexibility of the adaptor-binding domain (ABD) in p110α. Yet, the dimerization of PI3Kα is preserved through the ABD-p85α interaction. The local and global structural features induced by these mutations provide molecular insights into the activation of PI3Kα, deepen our understanding of the oncogenic mechanism of this important signaling molecule, and may facilitate the development of mutant-specific inhibitors.
    Keywords:  mass spectrometry; mutants; phosphoinositide 3-kinase (PI3K)
  2. EMBO J. 2022 Nov 10. e110833
      The AKT-mTOR pathway is a central regulator of cell growth and metabolism. Upon sustained mTOR activity, AKT activity is attenuated by a feedback loop that restrains upstream signaling. However, how cells control the signals that limit AKT activity is not fully understood. Here, we show that MASTL/Greatwall, a cell cycle kinase that supports mitosis by phosphorylating the PP2A/B55 inhibitors ENSA/ARPP19, inhibits PI3K-AKT activity by sustaining mTORC1- and S6K1-dependent phosphorylation of IRS1 and GRB10. Genetic depletion of MASTL results in an inefficient feedback loop and AKT hyperactivity. These defects are rescued by the expression of phosphomimetic ENSA/ARPP19 or inhibition of PP2A/B55 phosphatases. MASTL is directly phosphorylated by mTORC1, thereby limiting the PP2A/B55-dependent dephosphorylation of IRS1 and GRB10 downstream of mTORC1. Downregulation of MASTL results in increased glucose uptake in vitro and increased glucose tolerance in adult mice, suggesting the relevance of the MASTL-PP2A/B55 kinase-phosphatase module in controlling AKT and maintaining metabolic homeostasis.
    Keywords:  AKT; MASTL; cell cycle; glucose homeostasis; mTOR
  3. Front Immunol. 2022 ;13 982383
      Naive B cells use the chemokine receptor CXCR5 to enter B cell follicles, where they scan CXCL13-expressing ICAM-1<sup>+</sup> VCAM-1<sup>+</sup> follicular dendritic cells (FDCs) for the presence of antigen. CXCL13-CXCR5-mediated motility is mainly driven by the Rac guanine exchange factor DOCK2, which contains a binding domain for phosphoinositide-3,4,5-triphosphate (PIP3) and other phospholipids. While p110δ, the catalytic subunit of the class IA phosphoinositide-3-kinase (PI3K) δ, contributes to CXCR5-mediated B cell migration, the precise interdependency of DOCK2, p110δ, or other PI3K family members during this process remains incompletely understood. Here, we combined <i>in vitro</i> chemotaxis assays and <i>in vivo</i> imaging to examine the contribution of these two factors during murine naïve B cell migration to CXCL13. Our data confirm that p110δ is the main catalytic subunit mediating PI3K-dependent migration downstream CXCR5, whereas it does not contribute to chemotaxis triggered by CXCR4 or CCR7, two other chemokine receptors expressed on naïve B cells. The contribution of p110δ activity to CXCR5-driven migration was complementary to that of DOCK2, and pharmacological or genetic interference with both pathways completely abrogated B cell chemotaxis to CXCL13. Intravital microscopy of control and gene-deficient B cells migrating on FDCs confirmed that lack of DOCK2 caused a profound migration defect, whereas p110δ contributed to cell speed and directionality. B cells lacking active p110δ also displayed defective adhesion to ICAM-1; yet, their migration impairment was maintained on ICAM-1-deficient FDCs. In sum, our data uncover two complementary signaling pathways mediated by DOCK2 and p110δ, which enable CXCR5-driven naïve B cell examination of FDCs.
    Keywords:  B cell migration; CXCR5 (C-X-C motif chemokine receptor 5); DOCK2; intravital 2-photon microscopy; phosphoinoside-3-kinase
  4. Front Cell Dev Biol. 2022 ;10 1013001
      Recurrent missense mutations of the PIK3CA oncogene are among the most frequent drivers of human cancers. These often lead to constitutive activation of its product p110α, a phosphatidylinositol 3-kinase (PI3K) catalytic subunit. In addition to causing a broad range of cancers, the H1047R mutation is also found in affected tissues of a distinct set of congenital tumors and malformations. Collectively termed PIK3CA-related disorders (PRDs), these lead to overgrowth of brain, adipose, connective and musculoskeletal tissues and/or blood and lymphatic vessel components. Vascular malformations are frequently observed in PRD, due to cell-autonomous activation of PI3K signaling within endothelial cells. These, like most muscle, connective tissue and bone, are derived from the embryonic mesoderm. However, important organ systems affected in PRDs are neuroectodermal derivatives. To further examine their development, we drove the most common post-zygotic activating mutation of Pik3ca in neural crest and related embryonic lineages. Outcomes included macrocephaly, cleft secondary palate and more subtle skull anomalies. Surprisingly, Pik3ca-mutant subpopulations of neural crest origin were also associated with widespread cephalic vascular anomalies. Mesectodermal neural crest is a major source of non-endothelial connective tissue in the head, but not the body. To examine the response of vascular connective tissues of the body to constitutive Pik3ca activity during development, we expressed the mutation by way of an Egr2 (Krox20) Cre driver. Lineage tracing led us to observe new lineages that had normally once expressed Krox20 and that may be co-opted in pathogenesis, including vascular pericytes and perimysial fibroblasts. Finally, Schwann cell precursors having transcribed either Krox20 or Sox10 and induced to express constitutively active PI3K were associated with vascular and other tumors. These murine phenotypes may aid discovery of new candidate human PRDs affecting craniofacial and vascular smooth muscle development as well as the reciprocal paracrine signaling mechanisms leading to tissue overgrowth.
    Keywords:  PI3K; birth defect; cancer; cleft palate; embryo; neural crest; vascular anomaly
  5. Cancer Prev Res (Phila). 2022 Nov 07. pii: CAPR-22-0275. [Epub ahead of print]
      Antiestrogen medication is the only chemoprevention currently available for women at a high risk of developing breast cancer; however, antiestrogen therapy requires years to achieve efficacy and has adverse side effects. Therefore, it is important to develop an efficacious chemoprevention strategy that requires only a short-course of treatment. PIK3CA is commonly activated in breast atypical hyperplasia, the known precancerous precursor of breast cancer. Targeting PI3K signaling in these precancerous lesions may offer a new strategy for chemoprevention. Here, we first established a mouse model that mimics the progression from precancerous lesions to breast cancer. Next, we demonstrated that a short-course prophylactic treatment with the clinically approved PI3K inhibitor alpelisib slowed early lesion expansion and prevented cancer formation in this model. Furthermore, we showed that alpelisib suppressed ex vivo expansion of patient-derived atypical hyperplasia. Together, these data indicate that the progression of precancerous breast lesions heavily depends on the PI3K signaling, and that prophylactic targeting of PI3K activity can prevent breast cancer.
  6. Cells. 2022 Nov 07. pii: 3515. [Epub ahead of print]11(21):
      To ensure proper wound healing it is important to elucidate the signaling cues that coordinate leader and follower cell behavior to promote collective migration and proliferation for wound healing in response to injury. Using an ex vivo post-cataract surgery wound healing model we investigated the role of class I phosphatidylinositol-3-kinase (PI3K) isoforms in this process. Our findings revealed a specific role for p110α signaling independent of Akt for promoting the collective migration and proliferation of the epithelium for wound closure. In addition, we found an important role for p110α signaling in orchestrating proper polarized cytoskeletal organization within both leader and wounded epithelial follower cells to coordinate their function for wound healing. p110α was necessary to signal the formation and persistence of vimentin rich-lamellipodia extensions by leader cells and the reorganization of actomyosin into stress fibers along the basal domains of the wounded lens epithelial follower cells for movement. Together, our study reveals a critical role for p110α in the collective migration of an epithelium in response to wounding.
    Keywords:  PI3K; collective migration; follower cell; leader cell; p110α; wound healing
  7. Nat Commun. 2022 Nov 08. 13(1): 6744
      Targeting TEAD autopalmitoylation has been proposed as a therapeutic approach for YAP-dependent cancers. Here we show that TEAD palmitoylation inhibitor MGH-CP1 and analogues block cancer cell "stemness", organ overgrowth and tumor initiation in vitro and in vivo. MGH-CP1 sensitivity correlates significantly with YAP-dependency in a large panel of cancer cell lines. However, TEAD inhibition or YAP/TAZ knockdown leads to transient inhibition of cell cycle progression without inducing cell death, undermining their potential therapeutic utilities. We further reveal that TEAD inhibition or YAP/TAZ silencing leads to VGLL3-mediated transcriptional activation of SOX4/PI3K/AKT signaling axis, which contributes to cancer cell survival and confers therapeutic resistance to TEAD inhibitors. Consistently, combination of TEAD and AKT inhibitors exhibits strong synergy in inducing cancer cell death. Our work characterizes the therapeutic opportunities and limitations of TEAD palmitoylation inhibitors in cancers, and uncovers an intrinsic molecular mechanism, which confers potential therapeutic resistance.
  8. J Exp Med. 2023 Jan 02. pii: e20220342. [Epub ahead of print]220(1):
      Marginal zone (MZ) B cells represent innate-like B cells that mediate a fast immune response. The adhesion of MZ B cells to the marginal sinus of the spleen is governed by integrins. Here, we address the question of whether β1-integrin has additional functions by analyzing Itgb1fl/flCD21Cre mice in which the β1-integrin gene is deleted in mature B cells. We find that integrin β1-deficient mice have a defect in the differentiation of MZ B cells and plasma cells. We show that integrin β1-deficient transitional B cells, representing the precursors of MZ B cells, have enhanced B cell receptor (BCR) signaling, altered PI3K and Ras/ERK pathways, and an enhanced interaction of integrin-linked kinase (ILK) with the adaptor protein Grb2. Moreover, the MZ B cell defect of integrin β1-deficient mice could, at least in part, be restored by a pharmacological inhibition of the PI3K pathway. Thus, β1-integrin has an unexpected function in the differentiation and function of MZ B cells.
  9. Nat Commun. 2022 Nov 10. 13(1): 6808
      The mechanistic target of rapamycin complex 1 (mTORC1) integrates inputs from growth factors and nutrients, but how mTORC1 autoregulates its activity remains unclear. The MiT/TFE transcription factors are phosphorylated and inactivated by mTORC1 following lysosomal recruitment by RagC/D GTPases in response to amino acid stimulation. We find that starvation-induced lysosomal localization of the RagC/D GAP complex, FLCN:FNIP2, is markedly impaired in a mTORC1-sensitive manner in renal cells with TSC2 loss, resulting in unexpected TFEB hypophosphorylation and activation upon feeding. TFEB phosphorylation in TSC2-null renal cells is partially restored by destabilization of the lysosomal folliculin complex (LFC) induced by FLCN mutants and is fully rescued by forced lysosomal localization of the FLCN:FNIP2 dimer. Our data indicate that a negative feedback loop constrains amino acid-induced, FLCN:FNIP2-mediated RagC activity in renal cells with constitutive mTORC1 signaling, and the resulting MiT/TFE hyperactivation may drive oncogenesis with loss of the TSC2 tumor suppressor.
  10. Biochem J. 2022 Nov 11. 479(21): 2311-2325
      In the almost 70 years since the first hints of its existence, the phosphoinositide, phosphatidyl-D-myo-inositol 4,5-bisphosphate has been found to be central in the biological regulation of plasma membrane (PM) function. Here, we provide an overview of the signaling, transport and structural roles the lipid plays at the cell surface in animal cells. These include being substrate for second messenger generation, direct modulation of receptors, control of membrane traffic, regulation of ion channels and transporters, and modulation of the cytoskeleton and cell polarity. We conclude by re-evaluating PI(4,5)P2's designation as a signaling molecule, instead proposing a cofactor role, enabling PM-selective function for many proteins.
    Keywords:  5P2; PIP2; PtdIns4; lipid rafts; phospholipids; signaling
  11. Nat Commun. 2022 Nov 05. 13(1): 6700
      Lymphocystis disease virus-1 (LCDV-1) and several other Iridoviridae encode viral insulin/IGF-1 like peptides (VILPs) with high homology to human insulin and IGFs. Here we show that while single-chain (sc) and double-chain (dc) LCDV1-VILPs have very low affinity for the insulin receptor, scLCDV1-VILP has high affinity for IGF1R where it can antagonize human IGF-1 signaling, without altering insulin signaling. Consequently, scLCDV1-VILP inhibits IGF-1 induced cell proliferation and growth hormone/IGF-1 induced growth of mice in vivo. Cryo-electron microscopy reveals that scLCDV1-VILP engages IGF1R in a unique manner, inducing changes in IGF1R conformation that led to separation, rather than juxtaposition, of the transmembrane segments and hence inactivation of the receptor. Thus, scLCDV1-VILP is a natural peptide with specific antagonist properties on IGF1R signaling and may provide a new tool to guide development of hormonal analogues to treat cancers or metabolic disorders sensitive to IGF-1 without affecting glucose metabolism.
  12. Curr Opin Chem Biol. 2022 Oct 28. pii: S1367-5931(22)00109-0. [Epub ahead of print]71 102224
      Precise spatiotemporal organization and regulation of signal transduction networks are essential for cellular response to internal and external cues. To understand how this biochemical activity architecture impacts cellular function, many genetically encodable tools which regulate kinase activity at a subcellular level have been developed. In this review, we highlight various types of genetically encodable molecular tools, including tools to regulate endogenous kinase activity and biorthogonal techniques to perturb kinase activity. Finally, we emphasize the use of these tools alongside biosensors for kinase activity to measure and perturb kinase activity in real time for a better understanding of the cellular biochemical activity architecture.
    Keywords:  Biosensors; Cell signaling; Chemical biology; Chemical induced dimerization; Chemigenetic; Genetically encodable; Genetically encoded; Inhibitory peptides; Intrabodies; Kinase; Optogenetic; Spatiotemporal
  13. Int J Mol Sci. 2022 Oct 25. pii: 12876. [Epub ahead of print]23(21):
      PTEN has been implicated in the repair of DNA double-strand breaks (DSBs), particularly through homologous recombination (HR). However, other data fail to demonstrate a direct role of PTEN in DSB repair. Therefore, here, we report experiments designed to further investigate the role of PTEN in DSB repair. We emphasize the consequences of PTEN loss in the engagement of the four DSB repair pathways-classical non-homologous end-joining (c-NHEJ), HR, alternative end-joining (alt-EJ) and single strand annealing (SSA)-and analyze the resulting dynamic changes in their utilization. We quantitate the effect of PTEN knockdown on cell radiosensitivity to killing, as well as checkpoint responses in normal and tumor cell lines. We find that disruption of PTEN sensitizes cells to ionizing radiation (IR). This radiosensitization is associated with a reduction in RAD51 expression that compromises HR and causes a marked increase in SSA engagement, an error-prone DSB repair pathway, while alt-EJ and c-NHEJ remain unchanged after PTEN knockdown. The G2-checkpoint is partially suppressed after PTEN knockdown, corroborating the associated HR suppression. Notably, PTEN deficiency radiosensitizes cells to PARP inhibitors, Olaparib and BMN673. The results show the crucial role of PTEN in DSB repair and show a molecular link between PTEN and HR through the regulation of RAD51 expression. The expected benefit from combination treatment with Olaparib or BMN673 and IR shows that PTEN status may also be useful for patient stratification in clinical treatment protocols combining IR with PARP inhibitors.
    Keywords:  DDR; DSBs; HR; PARP inhibitors; PTEN; SSA; alt-EJ; c-NHEJ; ionizing radiation
  14. Cell Rep. 2022 Nov 08. pii: S2211-1247(22)01483-8. [Epub ahead of print]41(6): 111614
      Phosphatidylinositol 3-kinase catalytic subunit p110β is involved in tumorigenesis and hemostasis. However, it remains unclear if p110β also regulates platelet-mediated immune responses, which could have important consequences for immune modulation during anti-cancer treatment with p110β inhibitors. Thus, we investigate how platelet p110β affects inflammation and infection. Using a mouse model of Streptococcus pneumoniae-induced pneumonia, we find that both platelet-specific p110β deficiency and pharmacologic inhibition of p110β with TGX-221 exacerbate disease pathogenesis by preventing platelet-monocyte and neutrophil interactions, diminishing their infiltration and enhancing bacterial dissemination. Platelet p110β mediates neutrophil phagocytosis of S. pneumoniae in vitro and curtails bacteremia in vivo. Genetic deficiency or inhibition of platelet p110β also impairs macrophage recruitment in an independent model of sterile peritonitis. Our results demonstrate that platelet p110β dysfunction exacerbates pulmonary infection by impeding leukocyte functions. Thereby, our findings provide important insights into the immunomodulatory potential of PI3K inhibitors in bacterial infection.
    Keywords:  CP: Immunology; CP: Microbiology; PI3K; immunomodulation; infection; inflammation; leukocyte recruitment; p110 inhibitor; platelet; pneumonia; streptococcus
  15. Circ Res. 2022 Nov 07.
      BACKGROUND: As an integral component of cell membrane repair machinery, MG53 (mitsugumin 53) is important for cardioprotection induced by ischemia preconditioning and postconditioning. However, it also impairs insulin signaling via its E3 ligase activity-mediated ubiquitination-dependent degradation of IR (insulin receptor) and IRS1 (insulin receptor substrate 1) and its myokine function-induced allosteric blockage of IR. Here, we sought to develop MG53 into a cardioprotection therapy by separating its detrimental metabolic effects from beneficial actions.METHODS: Using immunoprecipitation-mass spectrometry, site-specific mutation, in vitro kinase assay, and in vivo animal studies, we investigated the role of MG53 phosphorylation at serine 255 (S255). In particular, utilizing recombinant proteins and gene knock-in approaches, we evaluated the potential therapeutic effect of MG53-S255A mutant in treating cardiac ischemia/reperfusion injury in diabetic mice.
    RESULTS: We identified S255 phosphorylation as a prerequisite for MG53 E3 ligase activity. Furthermore, MG53S255 phosphorylation was mediated by GSK3β (glycogen synthase kinase 3 beta) and markedly elevated in the animal models with metabolic disorders. Thus, IR-IRS1-GSK3β-MG53 formed a vicious cycle in the pathogenesis of metabolic disorders where aberrant insulin signaling led to hyper-activation of GSK3β, which in turn, phosphorylated MG53 and enhanced its E3 ligase activity, and further impaired insulin sensitivity. Importantly, S255A mutant eliminated the E3 ligase activity while retained cell protective function of MG53. Consequently, the S255A mutant, but not the wild type MG53, protected the heart against ischemia/reperfusion injury in db/db mice with advanced diabetes, although both elicited cardioprotection in normal mice. Moreover, in S255A knock-in mice, S255A mutant also mitigated ischemia/reperfusion-induced myocardial damage in the diabetic setting.
    CONCLUSIONS: S255 phosphorylation is a biased regulation of MG53 E3 ligase activity. The MG53-S255A mutant provides a promising approach for the treatment of acute myocardial injury, especially in patients with metabolic disorders.
    Keywords:  GSK3β; MG53; cardiac ischemia/reperfusion injury; cardioprotection; insulin resistance
  16. Nat Biotechnol. 2022 Nov 10.
      Cytosine base editors (CBEs) are larger and can suffer from higher off-target activity or lower on-target editing efficiency than current adenine base editors (ABEs). To develop a CBE that retains the small size, low off-target activity and high on-target activity of current ABEs, we evolved the highly active deoxyadenosine deaminase TadA-8e to perform cytidine deamination using phage-assisted continuous evolution. Evolved TadA cytidine deaminases contain mutations at DNA-binding residues that alter enzyme selectivity to strongly favor deoxycytidine over deoxyadenosine deamination. Compared to commonly used CBEs, TadA-derived cytosine base editors (TadCBEs) offer similar or higher on-target activity, smaller size and substantially lower Cas-independent DNA and RNA off-target editing activity. We also identified a TadA dual base editor (TadDE) that performs equally efficient cytosine and adenine base editing. TadCBEs support single or multiplexed base editing at therapeutically relevant genomic loci in primary human T cells and primary human hematopoietic stem and progenitor cells. TadCBEs expand the utility of CBEs for precision gene editing.
  17. Nat Commun. 2022 Nov 09. 13(1): 6782
      Germ-line hypomorphism of the pleiotropic transcription factor Myc in mice, either through Myc gene haploinsufficiency or deletion of Myc enhancers, delays onset of various cancers while mice remain viable and exhibit only relatively mild pathologies. Using a genetically engineered mouse model in which Myc expression may be systemically and reversibly hypomorphed at will, we asked whether this resistance to tumour progression is also emplaced when Myc hypomorphism is acutely imposed in adult mice. Indeed, adult Myc hypomorphism profoundly blocked KRasG12D-driven lung and pancreatic cancers, arresting their evolution at the early transition from indolent pre-tumour to invasive cancer. We show that such arrest is due to the incapacity of hypomorphic levels of Myc to drive release of signals that instruct the microenvironmental remodelling necessary to support invasive cancer. The cancer protection afforded by long-term adult imposition of Myc hypomorphism is accompanied by only mild collateral side effects, principally in haematopoiesis, but even these are circumvented if Myc hypomorphism is imposed metronomically whereas potent cancer protection is retained.
  18. Bioorg Chem. 2022 Oct 27. pii: S0045-2068(22)00617-4. [Epub ahead of print]130 106211
      Based on 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474), three series of novel 1,3,5-triazine or pyrimidine derivatives containing semicarbazones have been designed and synthesized to obtain new potent and selective PI3Kα inhibitors. Their inhibitory activities in vitro were evaluated against PI3Kα and three tumor-derived cell lines (U87-MG, MCF-7, and PC-3). We also tested promising compounds (A4, A6, A10, and B1) for other PI3K class I subtype (PI3Kβ, PI3Kδ, and PI3Kγ) activity. The representative compound A10 exhibited an IC50 value of 0.32 nM against PI3Kα, and demonstrated extraordinary subtype selectivity. Furthermore, compound A10 obviously inhibited proliferation of MCF-7 cell lines, induced a great decrease in mitochondrial membrane potential leading to apoptosis of cancer cells, and arrested G2 phase in a dose-dependent manner. Additionally, compound A10 induced significant tumor regressions in a xenograft mouse model of U87-MG cell line without an obvious sign of toxicity upon 20 mg/kg oral administration. Compound A10 may serve as a PI3Kα-selective inhibitor and provide the opportunity to spare patients the side effects associated with broader inhibition of the class I PI3K family.
    Keywords:  Antitumor; Kinase selectivity; PI3-kinase; Semicarbazones; Synthesis
  19. Cells. 2022 Oct 25. pii: 3358. [Epub ahead of print]11(21):
      Mammalian retromer complex contributes to multiple early endosome-associated trafficking pathways whose origins are dependent on which sorting nexin (SNX) they are complexed with. In an attempt to dissect out the contribution of individual retromer-SNX complexes, we examined the trafficking of EGFR in detail within a series of KO cell line models. We demonstrated that the depletion of retromer subunit Vps35 leads to decreased EGFR protein levels in resting cells with enhanced association of EGFR with lysosomal compartments. Compared to control cells, the addition of EGF to Vps35 KO cells resulted in a reduced rate of EGFR degradation; AKT activation and cell prolferation rates were elevated, while ERK activation remained relatively unchanged. These observations are consistent with a prolonged temporal association of EGFR within early endosomes due to the inefficiency of early endosome-associated protein trafficking pathways or organelle maturation due to retromer absence. We did not fully delineate the discrete contributions from retromer-associated SNXs to the phenotypes observed from retromer Vps35 depletion. While each of the knock-outs of SNX1/2, SNX3, or SNX27 promotes the enhanced association of EGFR with early endosomal compartments, only the decreased EGF-mediated EGFR degradation was observed in SNX1/2 dKO cells, while the enhanced AKT activation was only increased in SNX3 KO or SNX27 KO cells. Despite this, each of the knock-outs showed increased EGF-stimulated cell proliferation rates.
    Keywords:  EGF receptor; endosomes; protein trafficking; retromer
  20. Nat Commun. 2022 Nov 05. 13(1): 6681
      Transitioning from pluripotency to differentiated cell fates is fundamental to both embryonic development and adult tissue homeostasis. Improving our understanding of this transition would facilitate our ability to manipulate pluripotent cells into tissues for therapeutic use. Here, we show that membrane voltage (Vm) regulates the exit from pluripotency and the onset of germ layer differentiation in the embryo, a process that affects both gastrulation and left-right patterning. By examining candidate genes of congenital heart disease and heterotaxy, we identify KCNH6, a member of the ether-a-go-go class of potassium channels that hyperpolarizes the Vm and thus limits the activation of voltage gated calcium channels, lowering intracellular calcium. In pluripotent embryonic cells, depletion of kcnh6 leads to membrane depolarization, elevation of intracellular calcium levels, and the maintenance of a pluripotent state at the expense of differentiation into ectodermal and myogenic lineages. Using high-resolution temporal transcriptome analysis, we identify the gene regulatory networks downstream of membrane depolarization and calcium signaling and discover that inhibition of the mTOR pathway transitions the pluripotent cell to a differentiated fate. By manipulating Vm using a suite of tools, we establish a bioelectric pathway that regulates pluripotency in vertebrates, including human embryonic stem cells.
  21. Cell Syst. 2022 Oct 30. pii: S2405-4712(22)00430-6. [Epub ahead of print]
      The classic network of mitogen-activated protein kinases (MAPKs) is highly interconnected and controls a diverse array of biological processes. In multicellular eukaryotes, the MAPKs ERK, JNK, and p38 control opposing cell behaviors but are often activated simultaneously, raising questions about how input-output specificity is achieved. Here, we use multiplexed MAPK activity biosensors to investigate how cell fate control emerges from the connectivity and dynamics of the MAPK network. Through chemical and genetic perturbation, we systematically explore the outputs and functions of all the MAP3 kinases encoded in the human genome and show that MAP3Ks control cell fate by triggering unique combinations of MAPK activity. We show that these MAPK activity combinations explain the paradoxical dual role of JNK signaling as pro-apoptotic or pro-proliferative kinase. Overall, our integrative analysis indicates that the MAPK network operates as a unit to control cell fate and shifts the focus from MAPKs to MAP3Ks to better understand signaling-mediated control of cell fate.
    Keywords:  MAP kinase network; single-cell signaling dynamics
  22. Leukemia. 2022 Nov 09.
      Diffuse large B-cell lymphoma (DLBCL) is an aggressive disease that exhibits constitutive activation of phosphoinositide 3-kinase (PI3K) driven by chronic B-cell receptor signaling or PTEN deficiency. Since pan-PI3K inhibitors cause severe side effects, we investigated the anti-lymphoma efficacy of the specific PI3Kβ/δ inhibitor AZD8186. We identified a subset of DLBCL models within activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCL that were sensitive to AZD8186 treatment. On the molecular level, PI3Kβ/δ inhibition decreased the pro-survival NF-κB and AP-1 activity or led to downregulation of the oncogenic transcription factor MYC. In AZD8186-resistant models, we detected a feedback activation of the PI3K/AKT/mTOR pathway following PI3Kβ/δ inhibition, which limited AZD8186 efficacy. The combined treatment with AZD8186 and the mTOR inhibitor AZD2014 overcame resistance to PI3Kβ/δ inhibition and completely prevented outgrowth of lymphoma cells in vivo in cell line- and patient-derived xenograft mouse models. Collectively, our study reveals that subsets of DLBCLs are addicted to PI3Kβ/δ signaling and thus identifies a previously unappreciated role of the PI3Kβ isoform in DLBCL survival. Furthermore, our data demonstrate that combined targeting of PI3Kβ/δ and mTOR is effective in all major DLBCL subtypes supporting the evaluation of this strategy in a clinical trial setting.
  23. Nat Methods. 2022 Nov 07.
      We report the rational engineering of a remarkably stable yellow fluorescent protein (YFP), 'hyperfolder YFP' (hfYFP), that withstands chaotropic conditions that denature most biological structures within seconds, including superfolder green fluorescent protein (GFP). hfYFP contains no cysteines, is chloride insensitive and tolerates aldehyde and osmium tetroxide fixation better than common fluorescent proteins, enabling its use in expansion and electron microscopies. We solved crystal structures of hfYFP (to 1.7-Å resolution), a monomeric variant, monomeric hyperfolder YFP (1.6 Å) and an mGreenLantern mutant (1.2 Å), and then rationally engineered highly stable 405-nm-excitable GFPs, large Stokes shift (LSS) monomeric GFP (LSSmGFP) and LSSA12 from these structures. Lastly, we directly exploited the chemical stability of hfYFP and LSSmGFP by devising a fluorescence-assisted protein purification strategy enabling all steps of denaturing affinity chromatography to be visualized using ultraviolet or blue light. hfYFP and LSSmGFP represent a new generation of robustly stable fluorescent proteins developed for advanced biotechnological applications.
  24. ChemSystemsChem. 2022 Sep;pii: e202200011. [Epub ahead of print]4(5):
      Lipid rafts are ordered lipid domains that are enriched in saturated lipids, such as the ganglioside GM1. While lipid rafts are believed to exist in cells and to serve as signaling platforms through their enrichment in signaling components, they have not been directly observed in the plasma membrane without treatments that artificially cluster GM1 into large lattices. Here, we report that microscopic GM1-enriched domains can form, in the plasma membrane of live mammalian cells expressing the EphA2 receptor tyrosine kinase in response to its ligand ephrinA1-Fc. The GM1-enriched microdomains form concomitantly with EphA2-enriched microdomains. To gain insight into how plasma membrane heterogeneity controls signaling, we quantify the degree of EphA2 segregation and study initial EphA2 signaling steps in both EphA2-enriched and EphA2-depleted domains. By measuring dissociation constants, we demonstrate that the propensity of EphA2 to oligomerize is similar in EphA2-enriched and -depleted domains. However, surprisingly, EphA2 interacts preferentially with its downstream effector SRC in EphA2-depleted domains. The ability to induce microscopic GM1-enriched domains in live cells using a ligand for a transmembrane receptor will give us unprecedented opportunities to study the biophysical chemistry of lipid rafts.
    Keywords:  EphA2 receptor tyrosine kinase; lipid rafts; plasma membrane; protein-enriched domains; signaling platform
  25. J Clin Invest. 2022 Nov 10. pii: e162137. [Epub ahead of print]
      BACKGROUND: Acute febrile neutrophilic dermatosis (Sweet syndrome) is a potentially fatal multiorgan inflammatory disease characterized by fever, leukocytosis, and a rash with a neutrophilic infiltrate. Disease pathophysiology remains elusive, and current dogma suggests Sweet syndrome is a "reactive" process to an unknown antigen. Corticosteroids and steroid-sparing agents remain front-line therapies, but refractory cases pose a clinical challenge.METHODS: A 51-year-old woman with multiorgan Sweet syndrome developed serious corticosteroid-related side effects and was refractory to steroid-sparing agents. Blood counts, liver enzymes, and skin histopathology supported the diagnosis. Whole genome sequencing, transcriptomic profiling, and cellular assays of patient's skin and neutrophils were performed.
    RESULTS: We identified elevated IL-1 signaling in lesional Sweet syndrome skin caused by a PIK3R1 gain-of-function mutation specifically found in neutrophils. This mutation increased neutrophil migration towards IL-1β and neutrophil respiratory burst. Targeted treatment with an IL-1R1 antagonist in the patient resulted in a dramatic therapeutic response and enabled tapering of corticosteroids.
    CONCLUSIONS: Dysregulated PI3K-AKT signaling is the first signaling pathway linked to Sweet syndrome and suggests Sweet syndrome may be caused by acquired mutations that modulate neutrophil function. Moreover, integration of molecular data across multiple levels identified a distinct subtype within a heterogenous disease that resulted in a rational and successful clinical intervention. Future cases will benefit from efforts to identify potential mutations. The ability to directly interrogate diseased skin allows this method to be generalizable to other inflammatory diseases and demonstrates a potential personalized medicine approach for challenging patients.FUNDING Berstein Foundation, NIH, VA, Moseley Foundation, and H.T. Leung Foundation.
    Keywords:  Dermatology; Inflammation; Neutrophils; Signal transduction; Skin
  26. Nat Rev Genet. 2022 Nov 07.
      Programmable gene-editing tools have transformed the life sciences and have shown potential for the treatment of genetic disease. Among the CRISPR-Cas technologies that can currently make targeted DNA changes in mammalian cells, prime editors offer an unusual combination of versatility, specificity and precision. Prime editors do not require double-strand DNA breaks and can make virtually any substitution, small insertion and small deletion within the DNA of living cells. Prime editing minimally requires a programmable nickase fused to a polymerase enzyme, and an extended guide RNA that both specifies the target site and templates the desired genome edit. In this Review, we summarize prime editing strategies to generate programmed genomic changes, highlight their limitations and recent developments that circumvent some of these bottlenecks, and discuss applications and future directions.
  27. Nat Commun. 2022 Nov 08. 13(1): 6659
      DNA methylation undergoes dramatic age-related changes, first described more than four decades ago. Loss of DNA methylation within partially methylated domains (PMDs), late-replicating regions of the genome attached to the nuclear lamina, advances with age in normal tissues, and is further exacerbated in cancer. We present here experimental evidence that this DNA hypomethylation is directly driven by proliferation-associated DNA replication. Within PMDs, loss of DNA methylation at low-density CpGs in A:T-rich immediate context (PMD solo-WCGWs) tracks cumulative population doublings in primary cell culture. Cell cycle deceleration results in a proportional decrease in the rate of DNA hypomethylation. Blocking DNA replication via Mitomycin C treatment halts methylation loss. Loss of methylation continues unabated after TERT immortalization until finally reaching a severely hypomethylated equilibrium. Ambient oxygen culture conditions increases the rate of methylation loss compared to low-oxygen conditions, suggesting that some methylation loss may occur during unscheduled, oxidative damage repair-associated DNA synthesis. Finally, we present and validate a model to estimate the relative cumulative replicative histories of human cells, which we call "RepliTali" (Replication Times Accumulated in Lifetime).
  28. Front Oncol. 2022 ;12 1012391
      Background: It is critically important to study the real-world data of FDA-approved medications to understand the response rates and toxicities observed in the real-world population not represented in the clinical trials.Methods: We reviewed charts of patients diagnosed with metastatic, hormone receptor-positive, human epidermal growth factor receptor 2 negative, PIK3CA-mutated breast cancer treated with alpelisib from May 2019 to January 2022. Clinical characteristics and treatment outcomes were collected. The association of clinical characteristics with responses and adverse events (AEs) was evaluated using the logistic regression model.
    Results: 27 patients were included. Median age at alpelisib initiation 67 years (range: 44, 77 years). Majority of patients had excellent performance status at time of alpelisib initiation. Most patients had chronic comorbidities, notably; 2 patients had controlled type 2 diabetes mellitus at time of alpelisib initiation. Majority had a median of three lines of therapy (range: 1, 7) before alpelisib. Clinical responses were determined using RECIST v1.1. 3/27 (11.11%) patients discontinued therapy before response assessment due to grade 3 AEs. Overall response rate was 12.5% (3/24), with all partial responses (PR). The median duration of response was 5.77 months (range: 5.54, 8.98). 14/27 (51.9%) of patients required dose interruption/reduction. Overall, 23/27 (85.19%) patients discontinued alpelisib of which 11 (47.83%) discontinued alpelisib due to AEs. Median duration of treatment was 2 months in patients who had grade 3 AEs (range: <1.00, 8.30) and 6.28 (1.15, 10.43) in those who did not. Any grade AEs were reported in 24/27 (88.9%) patients, namely, hyperglycemia 16/27 (59.3%), nausea 11/27 (40.7%), diarrhea 10/27 (37.0%), fatigue 7/27 (25.9%) and rash 6/27 (22.2%). Grade 3 AEs were reported in 13/27 patients (50%), namely, hyperglycemia in 7/27 (53.8%) patients followed by skin rash 4/27 (30.8%), GI side effects 3/27 (23.1%). Those with progressive disease as best response to alpelisib, had more non-metabolic comorbidities, higher number of liver metastases, PIK3CA E545K mutations, and shorter duration on therapy compared to those with PR and stable disease.
    Conclusion: Patients should be counseled about the toxicity and modest benefit observed with alpelisib in real-world clinical practice when used in later lines of therapy.
    Keywords:  PIK3CA; adverse events; alpelisib; breast cancer; effectiveness; piqray; real-world