Methyl vinyl ketone and its analogs covalently modify PI3K and alter physiological functions by inhibiting PI3K signaling.

Reactive carbonyl species (RCS), which are abundant in the environment and are produced in vivo under stress, covalently bind to nucleophilic residues such as Cys in proteins. Disruption of protein function by RCS exposure is predicted to play a role in the development of various diseases such as cancer and metabolic disorders, but most studies on RCS have been limited to simple cytotoxicity validation, leaving their target proteins and resulting physiological changes unknown. In this study, we focused on methyl vinyl ketone (MVK), which is one of the main RCS found in cigarette smoke and exhaust gas. We found that MVK suppressed PI3K-Akt signaling, which regulates processes involved in cellular homeostasis, including cell proliferation, autophagy, and glucose metabolism. Interestingly, MVK inhibits the interaction between the epidermal growth factor receptor and PI3K. Cys656 in the SH2 domain of the PI3K p85 subunit, which is the covalently binding site of MVK, is important for this interaction. Suppression of PI3K-Akt signaling by MVK reversed epidermal growth factor-induced negative regulation of autophagy and attenuated glucose uptake. Furthermore, we analyzed the effects of the 23 RCS compounds with structures similar to MVK and showed that their analogs also suppressed PI3K-Akt signaling in a manner that correlated with their similarities to MVK. Our study demonstrates the mechanism of MVK and its analogs in suppressing PI3K-Akt signaling and modulating physiological functions, providing a model for future studies analyzing environmental reactive species.

Notch signaling regulates neural stem cell quiescence entry and exit in Drosophila.

Stem cells enter and exit quiescence as part of normal developmental programs and to maintain tissue homeostasis in adulthood. Although it is clear that stem cell intrinsic and extrinsic cues, local and systemic, regulate quiescence, it remains unclear whether intrinsic and extrinsic cues coordinate to control quiescence and how cue coordination is achieved. Here, we report that Notch signaling coordinates neuroblast intrinsic temporal programs with extrinsic nutrient cues to regulate quiescence in Drosophila. When Notch activity is reduced, quiescence is delayed or altogether bypassed, with some neuroblasts dividing continuously during the embryonic-to-larval transition. During embryogenesis before quiescence, neuroblasts express Notch and the Notch ligand Delta. After division, Delta is partitioned to adjacent GMC daughters where it transactivates Notch in neuroblasts. Over time, in response to intrinsic temporal cues and increasing numbers of Delta-expressing daughters, neuroblast Notch activity increases, leading to cell cycle exit and consequently, attenuation of Notch pathway activity. Quiescent neuroblasts have low to no active Notch, which is required for exit from quiescence in response to nutrient cues. Thus, Notch signaling coordinates proliferation versus quiescence decisions.

Discoidin domain receptor 2 signaling through PIK3C2α in fibroblasts promotes lung fibrosis.

Pulmonary fibrosis, especially idiopathic pulmonary fibrosis (IPF), portends significant morbidity and mortality, and current therapeutic options are suboptimal. We have previously shown that type I collagen signaling through discoidin domain receptor 2 (DDR2), a receptor tyrosine kinase expressed by fibroblasts, is critical for the regulation of fibroblast apoptosis and progressive fibrosis. However, the downstream signaling pathways for DDR2 remain poorly defined and could also be attractive potential targets for therapy. A recent phosphoproteomic approach indicated that PIK3C2α, a poorly studied member of the PI3 kinase family, could be a downstream mediator of DDR2 signaling. We hypothesized that collagen I/DDR2 signaling through PIK3C2α regulates fibroblast activity during progressive fibrosis. To test this hypothesis, we found that primary murine fibroblasts and IPF-derived fibroblasts stimulated with endogenous or exogenous type I collagen led to the formation of a DDR2/PIK3C2α complex, resulting in phosphorylation of PIK3C2α. Fibroblasts treated with an inhibitor of PIK3C2α or with deletion of PIK3C2α had fewer markers of activation after stimulation with TGFß and more apoptosis after stimulation with a Fas-activating antibody. Finally, mice with fibroblast-specific deletion of PIK3C2α had less fibrosis after bleomycin treatment than did littermate control mice with intact expression of PIK3Cα. Collectively, these data support the notion that collagen/DDR2/PIK3C2α signaling is critical for fibroblast function during progressive fibrosis, making this pathway a potential target for antifibrotic therapy. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Transient inhibition of neutrophil functions enhances the antitumor effect of intravenously delivered oncolytic vaccinia virus.

Oncolytic viruses (OVs) possess the unique ability to selectively replicate within tumor cells, leading to their destruction, while also reversing the immunosuppression within the tumor microenvironment and triggering an antitumor immune response. As a result, OVs have emerged as one of the most promising approaches in cancer therapy. However, the effective delivery of intravenously administered OVs faces significant challenges imposed by various immune cells within the peripheral blood, hindering their access to tumor sites. Notably, neutrophils, the predominant white blood cell population comprising approximately 50%-70% of circulating white cells in humans, show phagocytic properties. Our investigation revealed that the majority of oncolytic vaccinia viruses (VV) are engulfed and degraded by neutrophils in the bloodstream. The depletion of neutrophils using the anti-LY6G Ab (1-A8) resulted in an increased accumulation of circulating oncolytic VV in the peripheral blood and enhanced deposition at the tumor site, consequently amplifying the antitumor effect. Neutrophils heavily rely on PI3K signaling to sustain their phagocytic process. Additionally, our study determined that the inhibition of the PI3Kinase delta isoform by idelalisib (CAL-101) suppressed the uptake of oncolytic VV by neutrophils. This inhibition led to a greater presence of oncolytic VV in both the peripheral blood and at the tumor site, resulting in improved efficacy against the tumor. In conclusion, our study showed that inhibiting neutrophil functions can significantly enhance the antitumor efficacy of intravenous oncolytic VV.

K-Ras(V12) differentially affects the three Akt isoforms in lung and pancreatic carcinoma cells and upregulates E-cadherin and NCAM via Akt3.

K-Ras is the most frequently mutated Ras variant in pancreatic, colon and non-small cell lung adenocarcinoma. Activating mutations in K-Ras result in increased amounts of active Ras-GTP and subsequently a hyperactivation of effector proteins and downstream signaling pathways. Here, we demonstrate that oncogenic K-Ras(V12) regulates tumor cell migration by activating the phosphatidylinositol 3-kinases (PI3-K)/Akt pathway and induces the expression of E-cadherin and neural cell adhesion molecule (NCAM) by upregulation of Akt3. In vitro interaction and co-precipitation assays identified PI3-Kα as a bona fide effector of active K-Ras4B but not of H-Ras or N-Ras, resulting in enhanced Akt phosphorylation. Moreover, K-Ras(V12)-induced PI3-K/Akt activation enhanced migration in all analyzed cell lines. Interestingly, Western blot analyses with Akt isoform-specific antibodies as well as qPCR studies revealed, that the amount and the activity of Akt3 was markedly increased whereas the amount of Akt1 and Akt2 was downregulated in EGFP-K-Ras(V12)-expressing cell clones. To investigate the functional role of each Akt isoform and a possible crosstalk of the isoforms in more detail, each isoform was stably depleted in PANC-1 pancreatic and H23 lung carcinoma cells. Akt3, the least expressed Akt isoform in most cell lines, is especially upregulated and active in Akt2-depleted cells. Since expression of EGFP-K-Ras(V12) reduced E-cadherin-mediated cell-cell adhesion by induction of polysialylated NCAM, Akt3 was analyzed as regulator of E-cadherin and NCAM. Western blot analyses revealed pronounced reduction of E-cadherin and NCAM in the Akt3-kd cells, whereas Akt1 and Akt2 depletion upregulated E-cadherin, especially in H23 lung carcinoma cells. In summary, we identified oncogenic K-Ras4B as a key regulator of PI3-Kα-Akt signaling and Akt3 as a crucial regulator of K-Ras4B-induced modulation of E-cadherin and NCAM expression and localization.

Cell-permeable PI3 kinase competitive peptide inhibits KIT mutant mediated tumorigenesis of gastrointestinal stromal tumor (GIST).

BACKGROUND: Mutations in the receptor tyrosine kinase KIT are the main cause of gastrointestinal stromal tumor (GIST), and the KIT mutants mediated PI3 kinase activation plays a key role in the tumorigenesis of GIST. In this study, we aimed to block PI3 kinase activation by cell-permeable peptide and investigate its possible application in the treatment of GIST. METHODS AND RESULTS: We designed cell-permeable peptides based on the binding domain of PI3 kinase subunit p85 to KIT or PI3 kinase subunit p110, respectively, in order to compete for the binding between p85 and KIT or p110 and therefore inhibit the activation of PI3 kinases mediated by KIT. The results showed that the peptide can penetrate the cells, and inhibit the activation of PI3 kinases, leading to reduced cell survival and cell proliferation mediated by KIT mutants in vitro. Treatment of mice carrying germline KIT/V558A mutation, which can develop GIST, with the peptide that can compete for the binding between p85 and p110, led to reduced tumorigenesis of GIST. The peptide can further enhance the inhibition of the tumor growth by imatinib which is used as the first line targeted therapy of GIST. CONCLUSIONS: Our results showed that cell-permeable PI3 kinase competitive peptide can inhibit KIT-mediated PI3 kinase activation and tumorigenesis of GIST, providing a rationale to further test the peptide in the treatment of GIST and even other tumors with over-activation of PI3 kinases.

Acute Csk inhibition hinders B cell activation by constraining the PI3 kinase pathway.

T cell antigen receptor (TCR) and B cell antigen receptor (BCR) signaling are initiated and tightly regulated by Src-family kinases (SFKs). SFKs positively regulate TCR signaling in naïve T cells but have both positive and negative regulatory roles in BCR signaling in naïve B cells. The proper regulation of their activities depends on the opposing actions of receptor tyrosine phosphatases CD45 and CD148 and the cytoplasmic tyrosine kinase C-terminal Src kinase Csk. Csk is a major negative regulator of SFKs. Using a PP1-analog-sensitive Csk (CskAS) system, we have previously shown that inhibition of CskAS increases SFK activity, leading to augmentation of responses to weak TCR stimuli in T cells. However, the effects of Csk inhibition in B cells were not known. In this study, we surprisingly found that inhibition of CskAS led to marked inhibition of BCR-stimulated cytoplasmic free calcium increase and Erk activation despite increased SFK activation in B cells, contrasting the effects observed in T cells. Further investigation revealed that acute CskAS inhibition suppressed BCR-mediated phosphatidylinositol 3,4,5-trisphosphate (PIP3) production in B cells. Restoring PIP3 levels in B cells by CD19 cross-linking or SHIP1 deficiency eliminated the negative regulatory effect of CskAS inhibition. This reveals the critical role of Csk in maintaining an appropriate level of SFK activity and regulating PIP3 amounts as a means of compensating for SFK fluctuations to prevent inappropriate B cell activation. This regulatory mechanism controlling PIP3 amounts may also contribute to B cell anergy and self-tolerance.

GATOR1 Mutations Impair PI3 Kinase-Dependent Growth Factor Signaling Regulation of mTORC1.

GATOR1 (GAP Activity TOward Rag 1) is an evolutionarily conserved GTPase-activating protein complex that controls the activity of mTORC1 (mammalian Target Of Rapamycin Complex 1) in response to amino acid availability in cells. Genetic mutations in the GATOR1 subunits, NPRL2 (nitrogen permease regulator-like 2), NPRL3 (nitrogen permease regulator-like 3), and DEPDC5 (DEP domain containing 5), have been associated with epilepsy in humans; however, the specific effects of these mutations on GATOR1 function and mTORC1 regulation are not well understood. Herein, we report that epilepsy-linked mutations in the NPRL2 subunit of GATOR1, NPRL2-L105P, -T110S, and -D214H, increase basal mTORC1 signal transduction in cells. Notably, we show that NPRL2-L105P is a loss-of-function mutation that disrupts protein interactions with NPRL3 and DEPDC5, impairing GATOR1 complex assembly and resulting in high mTORC1 activity even under conditions of amino acid deprivation. Furthermore, our studies reveal that the GATOR1 complex is necessary for the rapid and robust inhibition of mTORC1 in response to growth factor withdrawal or pharmacological inhibition of phosphatidylinositol-3 kinase (PI3K). In the absence of the GATOR1 complex, cells are refractory to PI3K-dependent inhibition of mTORC1, permitting sustained translation and restricting the nuclear localization of TFEB, a transcription factor regulated by mTORC1. Collectively, our results show that epilepsy-linked mutations in NPRL2 can block GATOR1 complex assembly and restrict the appropriate regulation of mTORC1 by canonical PI3K-dependent growth factor signaling in the presence or absence of amino acids.

Zebrafish mutants in vegfab can affect endothelial cell proliferation without altering ERK phosphorylation and are phenocopied by loss of PI3K signaling.

The formation of appropriately patterned blood vessel networks requires endothelial cell migration and proliferation. Signaling through the Vascular Endothelial Growth Factor A (VEGFA) pathway is instrumental in coordinating these processes. mRNA splicing generates short (diffusible) and long (extracellular matrix bound) Vegfa isoforms. The differences between these isoforms in controlling cellular functions are not understood. In zebrafish, vegfaa generates short and long isoforms, while vegfab only generates long isoforms. We found that mutations in vegfaa had an impact on endothelial cell (EC) migration and proliferation. Surprisingly, mutations in vegfab more strongly affected EC proliferation in distinct blood vessels, such as intersegmental blood vessels in the zebrafish trunk and central arteries in the head. Analysis of downstream signaling pathways revealed no change in MAPK (ERK) activation, while inhibiting PI3 kinase signaling phenocopied vegfab mutant phenotypes in affected blood vessels. Together, these results suggest that extracellular matrix bound Vegfa might act through PI3K signaling to control EC proliferation in a distinct set of blood vessels during angiogenesis.

Assembly of nuclear dimers of PI3K regulatory subunits is regulated by the Cdc42-activated tyrosine kinase ACK.

Activated Cdc42-associated kinase (ACK) is an oncogenic nonreceptor tyrosine kinase associated with poor prognosis in several human cancers. ACK promotes proliferation, in part by contributing to the activation of Akt, the major effector of class 1A phosphoinositide 3-kinases (PI3Ks), which transduce signals via membrane phosphoinositol lipids. We now show that ACK also interacts with other key components of class 1A PI3K signaling, the PI3K regulatory subunits. We demonstrate ACK binds to all five PI3K regulatory subunit isoforms and directly phosphorylates p85α, p85ß, p50α, and p55α on Tyr607 (or analogous residues). We found that phosphorylation of p85ß promotes cell proliferation in HEK293T cells. We demonstrate that ACK interacts with p85α exclusively in nuclear-enriched cell fractions, where p85α phosphorylated at Tyr607 (pTyr607) also resides, and identify an interaction between pTyr607 and the N-terminal SH2 domain that supports dimerization of the regulatory subunits. We infer from this that ACK targets p110-independent p85 and further postulate that these regulatory subunit dimers undertake novel nuclear functions underpinning ACK activity. We conclude that these dimers represent a previously undescribed mode of regulation for the class1A PI3K regulatory subunits and potentially reveal additional avenues for therapeutic intervention.

Endothelial phosphoinositide 3-kinase-ß inactivation confers protection from immune-mediated vascular injury.

Heart transplant and recipient survival are limited by immune cell-mediated injury of the graft vasculature. We examined the role of the phosphoinositide 3-kinase-ß (PI3Kß) isoform in endothelial cells (EC) during coronary vascular immune injury and repair in mice. In minor histocompatibility-antigen mismatched allogeneic heart grafts, a robust immune response was mounted to each wild-type, PI3Kß inhibitor-treated, or endothelial-selective PI3Kß knockout (ECßKO) graft transplanted to wild-type recipients. However, microvascular EC loss and progressive occlusive vasculopathy only developed in control, but not PI3Kß-inactivated hearts. We observed a delay in inflammatory cell infiltration of the ECßKO grafts, particularly in the coronary arteries. Surprisingly, this was accompanied by an impaired display of proinflammatory chemokine and adhesion molecules by the ECßKO ECs. In vitro, tumor necrosis factor α-stimulated endothelial ICAM1 and VCAM1 expression was blocked by PI3Kß inhibition or RNA interference. Selective PI3Kß inhibition also blocked tumor necrosis factor α-stimulated degradation of inhibitor of nuclear factor kappa Bα and nuclear translocation of nuclear factor kappa B p65 in EC. These data identify PI3Kß as a therapeutic target to reduce vascular inflammation and injury.

Actin cytoskeleton remodeling disrupts physical barriers to infection and presents entry receptors to respiratory syncytial virus.

RSV is the leading cause of infant hospitalizations and a significant cause of paediatric and geriatric morbidity worldwide. Recently, we reported that insulin-like growth factor 1 receptor (IGF1R) was a receptor for respiratory syncytial virus (RSV) in airway epithelial cells and that activation of IGF1R recruited the coreceptor, nucleolin (NCL), to the cell surface. Cilia and mucus that line the airways pose a significant barrier to viral and bacterial infection. The cortical actin cytoskeleton has been shown by others to mediate RSV entry, so we studied the roles of the RSV receptors and actin remodelling during virus entry. We found that IGF1R expression and phosphorylation were associated with the ability of RSV to infect cells. Confocal immunofluorescence imaging showed that actin projections, a hallmark of macropinocytosis, formed around viral particles 30 min after infection. Consistent with prior reports we also found that virus particles were internalized into early endosome antigen-1 positive endosomes within 90 min. Inhibiting actin polymerization significantly reduced viral titre by approximately ten-fold. Inhibiting PI3 kinase and PKCζ in stratified air-liquid interface (ALI) models of the airway epithelium had similar effects on reducing the actin remodelling observed during infection and attenuating viral entry. Actin projections were associated with NCL interacting with RSV particles resting on apical cilia of the ALIs. We conclude that macropinocytosis-like actin projections protrude through normally protective cilia and mucus layers of stratified airway epithelium that helps present the IGF1R receptor and the NCL coreceptor to RSV particles waiting at the surface.

Triple-Negative Apocrine Carcinomas: Toward a Unified Group With Shared Molecular Features and Clinical Behavior.

Triple-negative apocrine carcinomas (TNACs) are rare breast tumors with limited studies evaluating their molecular characteristics and clinical behavior. We performed a histologic, immunohistochemical, genetic, and clinicopathologic assessment of 42 invasive TNACs (1 with a focal spindle cell component) from 41 patients, 2 pure apocrine ductal carcinomas in situ (A-DCIS), and 1 A-DCIS associated with spindle cell metaplastic carcinoma (SCMBC). All TNACs had characteristic apocrine morphology and expressed androgen receptor (42/42), gross cystic disease fluid protein 15 (24/24), and CK5/6 (16/16). GATA3 was positive in most cases (16/18, 89%), and SOX10 was negative (0/22). TRPS1 was weakly expressed in a minority of tumors (3/14, 21%). Most TNACs had low Ki67 proliferation (≤10% in 67%, 26/39), with a median index of 10%. Levels of tumor infiltrating lymphocytes were low (≤10% in 93%, 39/42, and 15% in 7%, 3/42). Eighteen percent of TNACs presented with axillary nodal metastasis (7/38). No patients treated with neoadjuvant chemotherapy achieved pathologic complete response (0%, 0/10). Nearly all patients with TNAC (97%, n = 32) were without evidence of disease at the time of study (mean follow-up of 62 months). Seventeen invasive TNACs and 10 A-DCIS (7 with paired invasive TNAC) were profiled by targeted capture-based next-generation DNA sequencing. Pathogenic mutations in phosphatidylinositol 3-kinase pathway genes PIK3CA (53%) and/or PIK3R1 (53%) were identified in all TNACs (100%), including 4 (24%) with comutated PTEN. Ras-MAPK pathway genes, including NF1 (24%), and TP53 were mutated in 6 tumors each (35%). All A-DCIS shared mutations, such as phosphatidylinositol 3-kinase aberrations and copy number alterations with paired invasive TNACs or SCMBC, and a subset of invasive carcinomas showed additional mutations in tumor suppressors (NF1, TP53, ARID2, and CDKN2A). Divergent genetic profiles between A-DCIS and invasive carcinoma were identified in 1 case. In summary, our findings support TNAC as a morphologically, immunohistochemically, and genetically homogeneous subgroup of triple-negative breast carcinomas and suggest overall favorable clinical behavior.

CCL6 promotes M2 polarization and inhibits macrophage autophagy by activating PI3-kinase/Akt signalling pathway during skin wound healing.

The transition of macrophages from the proinflammatory M1 to the anti-inflammatory M2 phenotype is crucial during the wound healing process. In this study, we assess the role of chemokine (C-C motif) ligand 6 (CCL6) in modulating macrophage polarization and wound healing. Initially, we observed significantly upregulated CCL6 expression in the skin tissue on the edge of the wound during the inflammation and proliferation phases. Furthermore, we discovered that the mice treated with rCCL6 had significantly accelerated wound healing and increased levels of M2-type macrophages. Using in vitro models, we found that CCL6 promotes the M2 polarization of macrophages by activating the PI3-kinase/Akt signalling pathway. Additionally, our results showed that CCL6 inhibited macrophage autophagy and accelerated wound healing, whereas the autophagy inducer rapamycin delayed wound healing. Finally, we determined that the PI3-kinase inhibitor LY294002 promoted macrophage autophagy and decreased M2 macrophages, indicating the importance of PI3-kinase in M2 polarization, and this process was reversed by CCL6. Taken together, our study demonstrates that CCL6 promotes M2 polarization, inhibits macrophage autophagy, and accelerates skin wound healing by activating the PI3-kinase/Akt signalling pathway.

Long-term treatment of cancer-prone germline PTEN mutant mice with low-dose rapamycin extends lifespan and delays tumour development.

PTEN is one of the most commonly inactivated tumour suppressor genes in sporadic cancer. Germline heterozygous PTEN gene alterations also underlie PTEN hamartoma tumour syndrome (PHTS), a rare human cancer-predisposition condition. A key feature of systemic PTEN deregulation is the inability to adequately dampen PI3-kinase (PI3K)/mTORC1 signalling. PI3K/mTORC1 pathway inhibitors such as rapamycin are therefore expected to neutralise the impact of PTEN loss, rendering this a more druggable context compared with 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 in, 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. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Based on 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474), design, synthesis and biological evaluation of novel PI3Kα selective inhibitors.

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 (PI3Kß, 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.

Structure-based drug design, synthesis, and biological evaluation of novel 1,3,5-triazine or pyrimidine derivatives containing benzoyl hydrazine moiety as PI3Kα selective inhibitors.

Phosphoinositide 3-kinase (PI3K) was an important cellular signal transducer, while PI3Kα was the most mutated family member in cancer. Selective PI3Kα inhibitors have become the frequent research in recent years because of their excellent curative effect and reduced side effects. Here, we described a series of PI3Kα inhibitors with 1,3,5-triazine or pyrimidine skeleton containing benzoyl hydrazine based on the pan-PI3K inhibitor ZSTK474 relying on the strategies of structure-based drug discovery (SBDD) and computer-aided drug design (CADD). Among them, compound F8 exhibited improved selective PI3Kα inhibition with an IC50 value of 0.14 nM and more significant anti-proliferative activities against three tumor-derived cell lines (PC-3 IC50 = 0.28 µM, HCT-116 IC50 = 0.57 µM, and U87-MG IC50 = 1.37 µM) than ZSTK-474. Compound F-8 induced a great decrease in mitochondrial membrane which caused cell cycle arrest at G1 phase and apoptosis in U87-MG cells in a dose-dependent manner. Furthermore, compound F8 induced significant tumor regressions in a xenograft mouse model of U87-MG cell line with no clear evidence of toxicity following intraperitoneal injection of 40 mg/kg. Compound F8 may serve as a PI3Kα-selective inhibitor and provided the opportunity to spare patients the side effects associated with broader inhibition of the class I PI3K family.

PI(4,5)P2 controls slit diaphragm formation and endocytosis in Drosophila nephrocytes.

Drosophila nephrocytes are an emerging model system for mammalian podocytes and proximal tubules as well as for the investigation of kidney diseases. Like podocytes, nephrocytes exhibit characteristics of epithelial cells, but the role of phospholipids in polarization of these cells is yet unclear. In epithelia, phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and phosphatidylinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) are asymmetrically distributed in the plasma membrane and determine apical-basal polarity. Here, we demonstrate that both phospholipids are present in the plasma membrane of nephrocytes, but only PI(4,5)P2 accumulates at slit diaphragms. Knockdown of Skittles, a phosphatidylinositol(4)phosphate 5-kinase, which produces PI(4,5)P2, abolished slit diaphragm formation and led to strongly reduced endocytosis. Notably, reduction in PI(3,4,5)P3 by overexpression of PTEN or expression of a dominant-negative phosphatidylinositol-3-kinase did not affect nephrocyte function, whereas enhanced formation of PI(3,4,5)P3 by constitutively active phosphatidylinositol-3-kinase resulted in strong slit diaphragm and endocytosis defects by ectopic activation of the Akt/mTOR pathway. Thus, PI(4,5)P2 but not PI(3,4,5)P3 is essential for slit diaphragm formation and nephrocyte function. However, PI(3,4,5)P3 has to be tightly controlled to ensure nephrocyte development.

The Amoebal Model for Macropinocytosis.

Macropinocytosis is a relatively unexplored form of large-scale endocytosis driven by the actin cytoskeleton. Dictyostelium amoebae form macropinosomes from cups extended from the plasma membrane, then digest their contents and absorb the nutrients in the endo-lysosomal system. They use macropinocytosis for feeding, maintaining a high rate of fluid uptake that makes assay and experimentation easy. Mutants collected over the years identify cytoskeletal and signalling proteins required for macropinocytosis. Cups are organized around plasma membrane domains of intense PIP3, Ras and Rac signalling, proper formation of which also depends on the RasGAPs NF1 and RGBARG, PTEN, the PIP3-regulated protein kinases Akt and SGK and their activators PDK1 and TORC2, Rho proteins, plus other components yet to be identified. This PIP3 domain directs dendritic actin polymerization to the extending lip of macropinocytic cups by recruiting a ring of the SCAR/WAVE complex around itself and thus activating the Arp2/3 complex. The dynamics of PIP3 domains are proposed to shape macropinocytic cups from start to finish. The role of the Ras-PI3-kinase module in organizing feeding structures in unicellular organisms most likely predates its adoption into growth factor signalling, suggesting an evolutionary origin for growth factor signalling.

Signaling Pathways that Regulate Macropinocytosis in Mammalian Cells.

Macropinocytosis is an evolutionarily conserved endocytic pathway that mediates non-selective uptake of extracellular fluid in bulk. Macropinocytosis is initiated by localized polymerization of the actin cytoskeleton, which generates plasma membrane protrusions that enclose part of the environment into large endocytic vesicles. From amoebae to mammalian cells, the actin dynamics that drive macropinosome formation are regulated by a conserved set of intracellular signaling proteins including Ras superfamily GTPases and PI3-kinases. In mammalian cells, multiple upstream signaling pathways control activity of these core regulators in response to cell-extrinsic and cell-intrinsic stimuli. Growth factor signaling pathways play a central role in macropinocytosis induction. In addition, an increasing number of functionally diverse processes has been identified as macropinocytosis regulators, including several nutrient-sensing and developmental signaling pathways. Many of these signaling pathways have proto-oncogenic properties, and their dysregulation drives the high macropinocytic activity that is commonly observed in cancer cells. These regulatory principles illustrate how macropinocytosis is controlled by complex upstream inputs to exert diverse cellular functions in physiological and pathological contexts.