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Background: Intrahepatic cholangiocarcinoma (ICC) is poorly treated due to the presence of an inhibitory immune microenvironment. Tumor-associated macrophages (TAM) are an important component of TME. ALOX5 is an important lipid metabolism enzyme in cancer progression, but the mechanism by which it regulates TAM to promote ICC progression is unknown. The aim of this study was to investigate the potential mechanism of TAM regulation by ALOX5 and the translational effect of targeting ALOX5. Methods: In this study, we investigated the association between the spatial localization of epithelial cells and TAMs by combining scRNA-seq analysis with multiplex immunofluorescence analysis. Through bulk sequencing analysis and spatial analysis, lipid metabolism genes closely related to TAM infiltration were screened. In vitro co-culture model was constructed to verify that ALOX5 and its downstream metabolite LTB4 promote M2 macrophage migration. Bulk sequencing after co-culture combined with single-cell analysis was performed to identify key pathways for up-regulation of M2 macrophage migration. Finally, the effect of CSF1R inhibitor (PLX3397) combined with ALOX5 inhibitor (Zileuton) in vivo was investigated by by xenograft tumor formation experiment in nude mice. Results: ALOX5 in ICC cells was a key lipid metabolism gene affecting the infiltration of M2 macrophages in TME. Mechanically, LTB4, a metabolite downstream of ALOX5, recruited M2 macrophages to migrate around tumor cells by binding to BLT1/BLT2 and activating the PI3K pathway, which ultimately lead to the promotion of ICC progression. Targeting CSF1R in combination with ALOX5 inhibitor effectively reduced tumor volume and M2 macrophage infiltration abundance. Conclusion: In ICC, LTB4, a metabolite secreted by ALOX5 of epithelial cells, binded to BLT1/BLT2 on TAM surface to activate PI3K pathway and promote TAM migration, thus promoting ICC progression. Targeting CSF1R in combination with ALOX5 inhibitor for ICC is a promising combination therapy modality. [ABSTRACT FROM AUTHOR]

Introduction: Increasingly, early‐stage non–small cell lung cancer (NSCLC) is treated with stereotactic body radiation therapy (SBRT). Although treatment is generally effective, a small subset of tumors will recur because of radioresistance. Preclinical studies suggested PI3K‐AKT‐mTOR activation mediates radioresistance. This study sought to validate this finding in tumor samples from patients who underwent SBRT for NSCLC. Methods: Patients with T1‐3N0 NSCLC treated with SBRT at our institution were included. Total RNA of formalin‐fixed paraffin‐embedded tumor biopsy specimens (pretherapy) was isolated and analyzed using the Clariom D assay. Risk scores from a PI3K activity signature and four published NSCLC signatures were generated and dichotomized by the median. Kaplan–Meier curves and Cox regressions were used to analyze their association with recurrence and overall survival (OS). The PI3K signature was also tested in a data set of resected NSCLC for additional validation. Results: A total of 92 patients were included, with a median follow‐up of 18.3 months for living patients. There was no association of any of the four published gene expression signatures with recurrence or OS. However, high PI3K risk score was associated with higher local recurrence (hazard ratio [HR], 11.72; 95% CI, 1.40–98.0; p =.023) and worse disease‐free survival (DFS) (HR, 3.98; 95% CI, 1.57–10.09; p =.0035), but not OS (p =.49), regional recurrence (p =.15), or distant recurrence (p =.85). In the resected NSCLC data set (n = 361), high PI3K risk score was associated with decreased OS (log‐rank p =.013) but not DFS (p = 0.54). Conclusions: This study validates that higher PI3K activity, measured by gene expression, is associated with local recurrence and worse DFS in early‐stage NSCLC patients treated with SBRT. This may be useful in prognostication and/or tailoring treatment, and merits further validation. In this single‐institution study of 92 patients with T1‐3 non–small cell lung cancer treated with stereotactic body radiation therapy, it was found that high PI3K activity, measured by gene expression using pretreatment biopsy specimens, was associated with worse local recurrence and disease‐free survival. [ABSTRACT FROM AUTHOR]

Sphingolipidoses are inherited metabolic disorders associated with glycosphingolipids accumulation, neurodegeneration, and neuroinflammation leading to severe neurological symptoms. Lysoglycosphingolipids (lysoGSLs), also known to accumulate in the tissues of sphingolipidosis patients, exhibit cytotoxicity. LysoGSLs are the possible pathogenic cause, but the mechanisms are still unknown in detail. Here, we first show that lysoGSLs are potential inhibitors of phosphoinositide 3‐kinase (PI3K) to reduce cell survival signaling. We found that phosphorylated Akt was commonly reduced in fibroblasts from patients with sphingolipidoses, including GM1/GM2 gangliosidoses and Gaucher's disease, suggesting the contribution of lysoGSLs to the pathogenesis. LysoGSLs caused cell death and decreased the level of phosphorylated Akt as in the patient fibroblasts. Extracellularly administered lysoGM1 permeated the cell membrane to diffusely distribute in the cytoplasm. LysoGM1 and lysoGM2 also inhibited the production of phosphatidylinositol‐(3,4,5)‐triphosphate and the translocation of Akt from the cytoplasm to the plasma membrane. We also predicted that lysoGSLs could directly bind to the catalytic domain of PI3K by in silico docking study, suggesting that lysoGSLs could inhibit PI3K by directly interacting with PI3K in the cytoplasm. Furthermore, we revealed that the increment of lysoGSLs amounts in the brain of sphingolipidosis model mice correlated with the neurodegenerative progression. Our findings suggest that the down‐regulation of PI3K/Akt signaling by direct interaction of lysoGSLs with PI3K in the brains is a neurodegenerative mechanism in sphingolipidoses. Moreover, we could propose the intracellular PI3K activation or inhibition of lysoGSLs biosynthesis as novel therapeutic approaches for sphingolipidoses because lysoGSLs should be cell death mediators by directly inhibiting PI3K, especially in neurons. [ABSTRACT FROM AUTHOR]

Polycystic ovary syndrome (PCOS) is a complicated gynecological endocrine disease that occurs in women of childbearing age. Protocatechuic acid is a phenol-rich compound derived from herbs and owns vital functions in numerous diseases. Howbeit, protocatechuic acid's impact on PCOS is unknown. A combination of in vivo and in vitro models was examined in this study. C57BL/6 mice were injected subcutaneously daily with dehydroepiandrosterone to establish a PCOS mouse model, and protocatechuic acid was intraperitoneally injected into PCOS mice. Granulosa cells of PCOS ovaries were also isolated. The function of protocatechuic acid was appraised using enzyme-linked immunosorbent assay, hematoxylin–eosin staining, reactive oxygen species (ROS) and LC3 levels analysis, flow cytometry, quantitative real-time PCR, and western blot. Meanwhile, the mechanism of protocatechuic acid was assessed with a series of molecular experiments. Protocatechuic acid owned no apparent toxic effect on mice. Functionally, protocatechuic acid owned a function of mitigating PCOS in vivo. Meanwhile, protocatechuic acid repressed ROS, autophagy, and apoptosis of PCOS ovarian granulosa cells in vitro. Mechanistically, rescue assays elucidated that the protective function of protocatechuic acid against PCOS was interrelated to the activation of the PI3K/AKT/mTOR axis. Protocatechuic acid alleviated PCOS symptoms in mice through PI3K signaling in granulosa cells to reduce ROS levels and apoptosis. [ABSTRACT FROM AUTHOR]

Rho proteins are part of the Ras superfamily, which function to modulate cytoskeletal dynamics including cell adhesion and motility. Recently, an activating mutation in Cdc42, a Rho family GTPase, was found in a patient sample of melanoma. Previously, our work had shown the PI3K was important downstream of mutationally active Cdc42. Our present study sought to determine whether PI3K was a crucial downstream partner for Cdc42 in a melanoma cells line with a BRAF mutation, which is the most common mutation in cutaneous melanoma. In this work we were able to show that Cdc42 contributes to proliferation, anchorage-independent growth, cell motility and invasion. Treatment with a pan-PI3K inhibitor was able to effectively ameliorate all these cancer phenotypes. These data suggest that PI3K may be an important target downstream of Cdc42 in melanoma. [ABSTRACT FROM AUTHOR]

Roginolisib (IOA-244) is a novel, non-ATP competitive phosphoinositide-3-kinase (PI3K) delta inhibitor that regulates Akt/mTOR signaling. Roginolisib was administered once daily to rats and dogs in dose-range finding (DRF) and 4-week GLP toxicology studies. Free plasma levels of roginolisib exceeded the cellular target engagement IC90 for PI3Kδ for ≥12 hours at doses of 5 mg/kg, the IC90 for PI3Kβ for ≥2 hours at doses ≥15 mg/kg, and the IC50 for PI3Kα for ≥2 hours at dose levels ≥45 mg/kg. Toxicity in rats occurred at doses ≥100 mg/kg. In dogs, we observed dose-dependent skin and gastrointestinal toxicity and doses ≥30 mg/kg had a greater incidence of mortality. Lymphoid tissue toxicity occurred in both species. Toxicities in dogs observed at the ≥15 mg/kg dose, affecting the digestive mucosa, liver, and skin, cleared after treatment cessation. Doses ≤75 mg/kg were tolerated in rats and the no-observed-adverse-effect-level (NOAEL) in rats was 15 mg/kg. Due to mainly epithelial lesions of the skin at 5 mg/kg and necrotizing damage of the intestinal epithelia at ≥15 mg/kg, no NOAEL was determined in dogs. However, the adverse effects observed in dogs at 5 mg/kg were considered monitorable and reversible in patients with advanced malignancies. Furthermore, the PK profile subsequently proved to be a decisive factor for achieving selective PI3Kδ inhibition without the toxicities observed in dogs. As the result of the unique PK profile of roginolisib, patients were able to take daily roginolisib without dose modification and showed pharmacodynamic PI3Kδ inhibition over several months without gastrointestinal or dermatologic toxicities. [ABSTRACT FROM AUTHOR]

Phosphoinositide 3‐kinase (PI3K) p110δ signalling negatively regulates the production of mouse IgE. However, there are disparities between the mouse and human IgE biology, and the role of PI3K p110δ in the production of human IgE is yet to be determined. To investigate the effect of PI3K p110δ inhibition in the production of human IgE we isolated human B cells from tonsil tissue and stimulated them with IL‐4 and anti‐CD40 antibody to induce class switching to IgE and IgG1 in the presence or absence of IC87114, a small molecule inhibitor of PI3K p110δ. Using FACS, RT‐PCR and ELISA we examined the effect of PI3K p110δ inhibition on IgE production and determined the mechanisms involved. Unlike in mice, we observed that PI3K p110δ inhibition significantly reduces the number of IgE+ switched cells and the amounts of secreted IgE in IL4 and anti‐CD40 cultures. However, the number of IgG1+ cells and secreted IgG1 were largely unaffected by PI3K p110δ inhibition. The expression levels of AID, ε and γ1 germinal transcripts or other factors involved in the regulation of CSR to IgE and IgG1 were also unaffected by IC87114. However, we found that IC87114 significantly decreases the proliferation of tonsil B cells stimulated with IL‐4 and anti‐CD40, specifically reducing the frequency of cells that had undergone 4 divisions or more. In addition, PI3K p110δ inhibition reduced the levels of IRF4 expression in IgE+ germinal centre‐like B cells leading to a block in plasma cell differentiation. In conclusion, PI3K p110δ signalling is required for the production of human IgE, which makes it a pharmacological target for the treatment of allergic disease. [ABSTRACT FROM AUTHOR]

Phosphoinositide 3-kinases (PI3K) and phosphoinositide 3-kinase-related protein kinases (PIKK) are two structurally related families of kinases that play vital roles in cell growth and DNA damage repair. Dysfunction of PIKK members and aberrant stimulation of the PI3K/AKT/mTOR signalling pathway are linked to a plethora of diseases including cancer. In recent decades, numerous inhibitors related to the PI3K/AKT/mTOR signalling have made great strides in cancer treatment, like copanlisib and sirolimus. Notably, most of the PIKK inhibitors (such as VX-970 and M3814) related to DNA damage response have also shown good efficacy in clinical trials. However, these drugs still require a suitable combination therapy to overcome drug resistance or improve antitumor activity. Based on the aforementioned facts, we summarised the efficacy of PIKK, PI3K, and AKT inhibitors in the therapy of human malignancies and the resistance mechanisms of targeted therapy, in order to provide deeper insights into cancer treatment. [ABSTRACT FROM AUTHOR]

Inhibition of PI3K pathway has become a desirable strategy for cancer treatment. In this work, a series of 2, 6, 8-substituted Imidazo[1,2-a]pyridine derivatives were designed and screened for their activities against PI3Kα and a panel of PI3Kα-addicted cancer cells. Among them, compound 35 was identified as a PI3Kα inhibitor with nanomolar potency as well as acceptable antiproliferative activity. Flow cytometry analysis confirmed 35 induced cell cycle arrest and apoptosis in T47D cells. In addition, it also showed desirable in vitro ADME properties. The design, synthesis, and SAR exploration of 35 are described within. [ABSTRACT FROM AUTHOR]

To explore the effect and mechanism of hydrogen sulfide (H2S) on autophagy and angiogenesis in skin wound of diabetic rats. METHODS: Among 36 healthy 8-week-old male Sprague-Dawley rats, 12 rats were selected as control group, and the remaining rats were intraperitoneally injected with streptozotocin (STZ) to induce diabetic model and were randomly divided into diabetes mellitus (DM) group and NaHS (H2S donor) intervention (DM+NaHS) group, with 12 rats in each group. A skin trauma model was established by excising the skin of the back of rats in each group. The rats in DM+NaHS group were intraperitoneally injected with NaHS (56 µmol/kg), and the rats in control and DM groups were daily received the same volume of normal saline for 21 consecutive days. The healing of skin wound was measured on days 0, 7, 14 and 21 after operation. On the 21st day after surgery, the content of H2S in skin tissues was detected by C-7Az fluorescent probe, and the morphological changes and angiogenesis of wound tissues were observed by HE staining. The expression of CD31 was detected by immunofluorescence staining, and endothelial autophagy was detected by double staining of CD31 and beclin-1. The protein levels of cystathionine γ-lyase (CSE), CD31, microtubule-associated protein 1 light chain 3 (LC3), beclin-1, P62, Bcl-2, Bax, phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB/Akt) and mammalian target of rapamycin (mTOR) in wound tissues were determined by Western blot. Caspase-3 and propidium iodide (PI) staining was used to detect cell apoptosis, and apoptosis of vascular endothelial cells was determined with CD31 and TUNEL double immunofluorescence staining. RESULTS: Compared with DM group, the wound healing rate, H2S content and CSE protein expression were significantly increased in DM+NaHS group (P<0. 01), but still lower than those in control group (P<0.01). HE staining showed that the wound surface in DM group was thin and wide, with few capillary, while that in DM+NaHS group was thicker with lots of capillary and wound width was reduced. Compared with DM group, CD31 expression was markedly increased (P<0. 01), the fluorescence intensity of caspase-3 and PI was significantly decreased (P<0. 01, and CD31+/beclin-1+ as well as CD31+/TUNEL+ cells were decreased (P<0. 01) in DM+NaHS group. Western blot analysis showed that compared with DM group, the levels of beclin-1, Bax and LC3-II/LC3-I were significantly decreased (P<0. 01), while the levels of P62 and Bcl-2, as well as ratios of p-PI3K/PI3K, p-Akt/ Akt and p-mTOR/mTOR were significantly increased (P<0. 01) in DM+NaHS group. CONCLUSION: H2S can promote skin wound healing, which may be related to activation of PI3K/Akt/mTOR signaling pathway, inhibition of endothelial autophagy and apoptosis, and promotion of angiogenesis in diabetic rats. [ABSTRACT FROM AUTHOR]

Skin mast cells (MCs) express high levels of MRGPRX2, FcεRI, and ST2, and vigorously respond to their ligands when triggered individually. IL-33/ST2 also potently synergizes with other receptors, but the molecular underpinnings are poorly understood. Human skin-derived MCs were stimulated via different receptors individually or jointly in the presence/absence of selective inhibitors. TNF was quantified by ELISA. Signaling cascades were studied by immunoblot. TNF was stimulated by FcεRI ≈ ST2 > MRGPRX2. Surprisingly, neither FcεRI nor MRGPRX2 stimulation elicited NF-κB activation (IκB degradation, p65 phosphorylation) in stark contrast to IL-33. Accordingly, TNF production did not depend on NF-κB in FcεRI- or MRGPRX2-stimulated MCs, but did well so downstream of ST2. Conversely, ERK1/2 and PI3K were the crucial modules upon FcεRI/MRGPRX2 stimulation, while p38 was key to the IL-33-elicited route. The different signaling prerequisites were mirrored by their activation patterns with potent pERK/pAKT after FcεRI/MRGPRX2, but preferential induction of pp38/NF-κB downstream of ST2. FcεRI/MRGPRX2 strongly synergized with IL-33, and some synergy was still observed upon inhibition of each module (ERK1/2, JNK, p38, PI3K, NF-κB). IL-33's contribution to synergism was owed to p38 > JNK > NF-κB, while the partner receptor contributed through ERK > PI3K ≈ JNK. Concurrent IL-33 led to slightly prolonged pERK (downstream of MRGPRX2) or pAKT (activated by FcεRI), while the IL-33-elicited modules (pp38/NF-κB) remained unaffected by co-stimulation of FcεRI/MRGPRX2. Collectively, the strong synergistic activity of IL-33 primarily results from the complementation of highly distinct modules following co-activation of the partner receptor rather than by altered signal strength of the same modules. [ABSTRACT FROM AUTHOR]

Background & Aims: The class I‐ phosphatidylinositol‐3 kinases (PI3Ks) signalling is dysregulated in almost all human cancers whereas the isoform‐specific roles remain poorly investigated. We reported that the isoform δ (PI3Kδ) regulated epithelial cell polarity and plasticity and recent developments have heightened its role in hepatocellular carcinoma (HCC) and solid tumour progression. However, its role in cholangiocarcinoma (CCA) still lacks investigation. Approach & Results: Immunohistochemical analyses of CCA samples reveal a high expression of PI3Kδ in the less differentiated CCA. The RT‐qPCR and immunoblot analyses performed on CCA cells stably overexpressing PI3Kδ using lentiviral construction reveal an increase of mesenchymal and stem cell markers and the pluripotency transcription factors. CCA cells stably overexpressing PI3Kδ cultured in 3D culture display a thick layer of ECM at the basement membrane and a wide single lumen compared to control cells. Similar data are observed in vivo, in xenografted tumours established with PI3Kδ‐overexpressing CCA cells in immunodeficient mice. The expression of mesenchymal and stemness genes also increases and tumour tissue displays necrosis and fibrosis, along with a prominent angiogenesis and lymphangiogenesis, as in mice liver of AAV8‐based‐PI3Kδ overexpression. These PI3Kδ‐mediated cell morphogenesis and stroma remodelling were dependent on TGFβ/Src/Notch signalling. Whole transcriptome analysis of PI3Kδ using the cancer cell line encyclopedia allows the classification of CCA cells according to cancer progression. Conclusions: Overall, our results support the critical role of PI3Kδ in the progression and aggressiveness of CCA via TGFβ/src/Notch‐dependent mechanisms and open new directions for the classification and treatment of CCA patients. [ABSTRACT FROM AUTHOR]

The δ isoform of class I PI3K (PI3Kδ) has been shown as a promising target for the treatment of hematologic malignancies and immune diseases. Herein, a series of pyrido[3,2‐d]pyrimidine derivatives were designed, synthesized and evaluated for the preliminary bioactivity. Compared with idelalisib, compound S5 exhibited excellent enzyme activity against PI3Kδ (IC50 = 2.82 nM) and strong antiproliferation activity against SU‐DHL‐6 cells (IC50 = 0.035 μM). Besides, S5 inhibited the phosphorylation of Akt, which is downstream of PI3Kδ, in concentration‐dependent manner. In view of the significant improvement in potency of PI3Kδ and selectivity over other PI3K isoforms, Compound S5 deserved further investigation as a promising PI3Kδ inhibitor. [ABSTRACT FROM AUTHOR]

Pouzolzia zeylanica (L.) Benn. is a Chinese herbal medicine widely used for its anti‐inflammatory and pus‐removal properties. To explore its potential anti‐inflammatory mechanism, quercetin 3,7‐dirhamnoside (QDR), the main flavonoid component of P. zeylanica (L.) Benn., was extracted and purified. The potential anti‐inflammatory targets of QDR were predicted using network analysis. These potential targets were verified using molecular docking, molecular dynamics simulations, and in vitro experiments. Consequently, 342 potential anti‐inflammatory QDR targets were identified. By analyzing the intersection between the protein–protein interaction and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, we identified several potential protein targets of QDR, including RAC‐alpha serine/threonine‐protein kinase (AKT1), Ras‐related C3 botulinum toxin substrate 1 (RAC1), nitric oxide synthase 3 (NOS3), serine/threonine‐protein kinase mTOR (mTOR), epidermal growth factor receptor (EGFR), growth factor receptor‐bound protein 2 (GRB2), and endothelin‐1 receptor (EDNRA). QDR has anti‐inflammatory activity and regulates immune responses and apoptosis through chemokines, Phosphatidylinositol 3‐kinase 3(PI3K)/AKT, cAMP, T‐cell receptor, and Ras signaling pathways. Molecular docking analysis showed that QDR has good binding abilities with AKT1, mTOR, and NOS3. In addition, molecular dynamics simulations demonstrated that the protein–ligand complex systems formed between QDR and AKT1, mTOR, and NOS3 have high dynamic stability, and their protein–ligand complex systems possess strong binding ability. In RAW264.7 macrophages, QDR significantly inhibited lipopolysaccharides (LPS)‐induced inducible nitric oxide synthase expression, nitric oxide (NO) release and the generation of proinflammatory cytokines IL‐6, IL‐1β, and TNF‐α. QDR downregulated the expression of p‐AKT1(Ser473)/AKT1 and p‐mTOR (Ser2448)/mTOR, and upregulated the expression of NOS3, Rictor, and Raptor. This indicates that the anti‐inflammatory mechanisms of QDR involve regulation of AKT1 and mTOR to prevent apoptosis and of NOS3 which leads to the release of endothelial NO. Thus, our study elucidated the potential anti‐inflammatory mechanism of QDR, the main flavonoid found in P. zeylanica (L.) Benn. [ABSTRACT FROM AUTHOR]

Background: Acute myocardial infarction (AMI) is one of the most important causes of mortality among patients with cardiovascular disease. Ginsenoside Rh2 plays a protective role in cardiovascular diseases. Furthermore, pyroptosis reportedly participates in regulating the occurrence and development of AMI. However, whether ginsenoside Rh2 contributes to mitigating AMI by regulating cardiomyocyte pyroptosis remains unknown. Methods: In the present study, we established an AMI model in rats. Next, we determined the effects of ginsenoside Rh2 on AMI by examining the myocardial infarct area, while regulation of myocardial pyroptosis was determined by assessing related factors. We established a cardiomyocyte model using hypoxia/reoxygenation (H/R) treatment. The expression of pyroptosis-related factors was determined following ginsenoside Rh2 treatment. In addition, we evaluated the correlation between ginsenoside Rh2 and the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway at the mechanistic level. Results: Herein, we observed that ginsenoside Rh2 alleviated AMI in rats and cells. Notably, the expression levels of inflammatory factors were reduced in AMI rats and cells. Furthermore, AMI rats and cells exhibited high expression levels of cleaved caspase-1 and gasdermin D, which were downregulated following treatment with ginsenoside Rh2. Further analysis revealed that ginsenoside Rh2 could inhibit cardiomyocyte pyroptosis by regulating the PI3K/AKT signaling pathway. Conclusions: Collectively, the findings of the present study demonstrated that ginsenoside Rh2 regulates pyroptosis in cardiomyocytes to alleviate AMI in vivo and in vitro, thereby affording a novel therapeutic approach to treat AMI. [ABSTRACT FROM AUTHOR]

A critical need in the field of genotype-matched targeted therapy in cancer is to identify patients unlikely to respond to precision medicines. This will manage expectations of individualised therapies and avoid clinical progression to a point where institution of alternative treatments might not be possible. We examined the evidence base of the impact of genomic context on which targeted alterations are inscribed to identify baseline biomarkers distinguishing those obtaining the expected response from those with less benefit from targeted therapies. A comprehensive narrative review was conducted: scoping searches were undertaken in PubMed, Cochrane Database of Systematic Reviews, and PROSPERO. Outcomes included in meta-analysis were progression-free and overall survival. Data were extracted from Kaplan–Meier and used to calculate hazard ratios. Studies presenting data on two molecular subcohorts (e.g. co-mutation versus no co-mutation) were included in fixed meta-analysis. Other studies were used for descriptive purposes. The presence of concomitant driver mutations, higher tumour mutational burden (TMB), greater copy number burden, and APOBEC signatures significantly reduces benefits of targeted therapy in lung cancers in never smokers (LCINS - less than 100 cigarettes per lifetime) and breast cancer, cancers with low TMB. LCINS have significantly poorer outcomes if their cancers harbour p53 co-mutations, an effect also seen in human epidermal growth factor receptor 2-positive (HER2+) breast cancer patients (trastuzumab) and head and neck cancer patients [phosphoinositide 3-kinase (PI3K) inhibition]. PI3K co-alterations have less impact when targeting epidermal growth factor receptor mutations and anaplastic lymphoma kinase fusions, but significantly reduce the impact of targeting HER2 and MET amplifications. SMARCA4 co-mutations predict for poor outcome in patients treated with osimertinib and sotorasib. In BRAF-mutant melanoma, whilst there are no genomic features distinguishing exceptional responders from primary progressors, there are clear transcriptomic features dichotomising these outcomes. To our knowledge, this is the most comprehensive review to date of the impact of genomic context on outcomes with targeted therapy. It represents a valuable resource informing progress towards contextualised precision medicine. • Concomitant driver mutations, TMB, copy number, and APOBEC impact targeted therapy outcomes in LCINS and breast cancer. • Several cancers have poorer outcomes if they harbour concomitant p53 mutations. • Concomitant PI3K alterations have less impact when targeting EGFR and ALK, but reduce the impact of targeting HER2 and MET. • In metastatic BRAF-mutant melanoma patients there are clear transcriptomic signatures determining diverse outcomes. • This review represents a valuable resource informing progress towards a more contextualised precision medicine. [ABSTRACT FROM AUTHOR]

ER‐phagy is a selective autophagy process that targets specific regions of the endoplasmic reticulum (ER) for removal via lysosomal degradation. During cellular stress induced by starvation, cargo receptors concentrate at distinct ER‐phagy sites (ERPHS) to recruit core autophagy proteins and initiate ER‐phagy. However, the molecular mechanism responsible for ERPHS formation remains unclear. In our study, we discovered that the autophagy regulator UV radiation Resistance‐Associated Gene (UVRAG) plays a crucial role in orchestrating the assembly of ERPHS. Upon starvation, UVRAG localizes to ERPHS and interacts with specific ER‐phagy cargo receptors, such as FAM134B, ATL3, and RTN3L. UVRAG regulates the oligomerization of cargo receptors and facilitates the recruitment of Atg8 family proteins. Consequently, UVRAG promotes efficient ERPHS assembly and turnover of both ER sheets and tubules. Importantly, UVRAG‐mediated ER‐phagy contributes to the clearance of pathogenic proinsulin aggregates. Remarkably, the involvement of UVRAG in ER‐phagy initiation is independent of its canonical function as a subunit of class III phosphatidylinositol 3‐kinase complex II. Synopsis: ER‐phagy initiation is localized to distinct sites on the ER, but the assembly mechanism of these ER‐phagy sites (ERPHS) remains unclear. This study identifies UVRAG as a novel regulator of ERPHS formation, operating independently to its canonical function in PI3KC3 complex II. UVRAG translocates to ERPHS and interacts with diverse ER‐phagy cargo receptors, such as FAM134B, ATL3, and RTN3L.UVRAG promotes oligomerization of ER‐phagy cargo receptors.UVRAG enhances the binding of ER‐phagy cargo receptors to Atg8 family proteins.UVRAG‐mediated ER‐phagy promotes the degradation of ER sheets, tubules, and aggregation‐prone ER luminal proteins, such as pathogenic proinsulin. [ABSTRACT FROM AUTHOR]

Resveratrol is a natural polyphenol found in grapes and beneficial for human health. Resveratrol regulates basic fibroblast growth factor (bFGF)-induced osteoprotegerin synthesis through Akt pathway in osteoblast-like MC3T3-E1 cells. In this study, we investigated resveratrol effects on bFGF-induced macrophage colony-stimulating factor (M-CSF) synthesis in MC3T3-E1 cells. bFGF significantly stimulated release and mRNA expression of M-CSF, which was reduced by resveratrol and SRT1720, sirtuin 1 (SIRT1) activator. Inauhzin, SIRT1 inhibitor, reversed inhibitory effects of resveratrol on bFGF-induced mRNA expression of M-CSF. Deguelin, Akt inhibitor, and LY294002, phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, reduced bFGF-induced M-CSF synthesis. Inauhzin reversed inhibitory effects of resveratrol on bFGF-induced Akt phosphorylation. Suppressive effect of resveratrol on bFGF-induced osteoprotegerin mRNA expression was confirmed in the identical samples using in experiment of M-CSF mRNA expression. Therefore, resveratrol reduces bFGF-induced M-CSF synthesis in addition to osteoprotegerin synthesis by inhibiting PI3-kinase/Akt pathway and suppressive effects are mediated through SIRT1 activation in osteoblasts. [ABSTRACT FROM AUTHOR]

Reproduction is characterized by a series of massive renovations at molecular, cellular, and tissue levels. Recent studies have strongly tended to reveal the involvement of basic molecular pathways such as autophagy, a highly conserved eukaryotic cellular recycling, during reproductive processes. This review comprehensively describes the current knowledge, updated to September 2022, of autophagy contribution during reproductive processes in males including spermatogenesis, sperm motility and viability, and male sex hormones and females including germ cells and oocytes viability, ovulation, implantation, fertilization, and female sex hormones. Furthermore, the consequences of disruption in autophagic flux on the reproductive disorders including oligospermia, azoospermia, asthenozoospermia, teratozoospermia, globozoospermia, premature ovarian insufficiency, polycystic ovarian syndrome, endometriosis, and other disorders related to infertility are discussed as well. Abbreviations: AKT/protein kinase B: AKT serine/threonine kinase; AMPK: AMP-activated protein kinase; ATG: autophagy related; E2: estrogen; EDs: endocrine disruptors; ER: endoplasmic reticulum; FSH: follicle stimulating hormone; FOX: forkhead box; GCs: granulosa cells; HIF: hypoxia inducible factor; IVF: in vitro fertilization; IVM: in vitro maturation; LCs: Leydig cells; LDs: lipid droplets; LH: luteinizing hormone; LRWD1: leucine rich repeats and WD repeat domain containing 1; MAP1LC3: microtubule associated protein 1 light chain 3; MAPK: mitogen-activated protein kinase; MTOR: mechanistic target of rapamycin kinase; NFKB/NF-kB: nuclear factor kappa B; P4: progesterone; PCOS: polycystic ovarian syndrome; PDLIM1: PDZ and LIM domain 1; PI3K: phosphoinositide 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns3K: class III phosphatidylinositol 3-kinase; POI: premature ovarian insufficiency; ROS: reactive oxygen species; SCs: Sertoli cells; SQSTM1/p62: sequestosome 1; TSGA10: testis specific 10; TST: testosterone; VCP: vasolin containing protein. [ABSTRACT FROM AUTHOR]

Objective: Acute myeloid leukemia (AML) is a malignant disease characterized by the uncontrolled growth, differentiation, and proliferation of immature hematopoietic cells. Patients with AML often have poor survival rates, which are associated with specific gene mutations in FLT3, CEBPA, and NPM1. The phosphatidylinositol 3-kinase (PI3K) pathway, a lipase pathway, is activated in many malignancies, including AML. Given the low survival rates in AML, this study identified candidate drugs that could inhibit the PI3K pathway, thereby offering a potential treatment for AML, by using a drug-repurposing approach. Materials and Methods: Online bioinformatics tools were utilized to identify pathway-related genes and FDA-approved drugs. Subsequently, molecular docking was performed to determine the binding affinity values. Important genes were identified by evaluating their impact on survival and their aberrant expression in the tumor. In this study, genes such as VAV1, GSK3B, MTOR, PDPK1, PRR5, TSC2, AKT3, and CREB1 were determined and docked with their potential inhibitors. Particular attention was paid to VAV1 because there were no known potential VAV1 inhibitors used in AML. Results: The docking results were ranked, and the proposed gene-drug pairs were identified as tideglusib and fostamatinib for the inhibition of GSK3B, pimecrolimus and fostamatinib for the inhibition of MTOR, and fostamatinib for the inhibition of PDPK1. Furthermore, nebivolol, darifenacin, dihydroergotamine, libanserin and entereg were identified as potential inhibitors of VAV1 in AML. Conclusion: To sum up, most effective gene-drug pairs according to binding affinities were proposed as candidate inhibitor drugs for AML. [ABSTRACT FROM AUTHOR]

Background. Thyroid-associated orbitopathy (TAO) is a disease associated with autoimmune thyroid disorders and it can lead to proptosis, diplopia, and vision-threatening compressive optic neuropathy. To comprehensively understand the molecular mechanisms underlying orbital adipogenesis in TAO, we characterize the intrinsic molecular properties of orbital adipose/connective tissue from patients with TAO and control individuals. Methods. RNA sequencing analysis (RNA-seq) was performed to measure the gene expression of orbital adipose/connective tissues of TAO patients. Differentially expressed genes (DEGs) were detected and analyzed through Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and Gene Set Enrichment Analysis (GSEA). The protein-protein interaction (PPI) network was constructed using the STRING database, and hub genes were identified by the Cytoscape plug-in, cytoHubba. We validated several top DEGs through quantitative real-time polymerase chain reaction (qRT-PCR). Results. We identified 183 DEGs in adipose tissue between TAO patients (nD3) and control patients (nD3) through RNA sequencing, including 114 upregulated genes and 69 downregulated genes. The PPI network of these DEGs had 202 nodes and 743 edges. PCR-based validation results of orbital adipose tissue showed multiple top-ranked genes in TAO patients (n = 4) are immune and inflammatory response genes compared with the control individual (n = 4). They include ceruloplasmin isoform x3 (CP), alkaline tissue-nonspecific isozyme isoform x1 (ALPL), and angiotensinogen (AGT), which were overrepresented by 2.27-to 6.40-fold. Meanwhile, protein mab-21-like 1 (MAB21L1), phosphoinositide 3-kinase gamma-subunit (PIK3C2G), and clavesin-2 (CLVS2) decreased by 2.6% to 32.8%. R-spondin 1 (RSPO1), which is related to oogonia differentiation and developmental angiogenesis, was significantly downregulated in the orbital muscle tissues of patients with TAO compared with the control groups (P D0:024). Conclusions. Our results suggest that there are genetic differences in orbital adipose-connective tissues derived from TAO patients. The upregulation of the inflammatory response in orbital fat of TAO may be consistent with the clinical phenotype like eyelid edema, exophthalmos, and excess tearing. Downregulation of MAB21L1, PIK3C2G, and CLVS2 in TAO tissue demonstrates dysregulation of differentiation, oxidative stress, and developmental pathways. [ABSTRACT FROM AUTHOR]

A growing concern of cardiotoxicity induced by PI3K inhibitors has raised the requirements to evaluate the structure-cardiotoxicity relationship (SCR) in the development process of novel inhibitors. Based on three bioisosteric 7-azaindazole-based candidate inhibitors namely FD269, FD268 and FD274 that give same order of inhibitory concentration 50% (IC50) magnitude against PI3Ks, in this work, we proposed to systematically evaluate the SCR of 7-azaindazole-based PI3K inhibitors designed by bioisosteric approach. The 24-h lethal concentrations 50% (LC50) of FD269, FD268 and FD274 against zebrafish embryos were 0.35, 4.82 and above 50 μM (not detected), respectively. Determination of the heart rate, pericardial and yolk-sac areas and vascular malformation confirmed the remarkable reduction in the cardiotoxicity of from FD269 to FD268 and to FD274. The IC50s of all three compounds against the hERG channel were tested on the CHO cell line that constitutively expressing hERG channel, which were all higher than 20 μM. The transcriptomic analysis revealed that FD269 and FD268 induced the up-regulation of noxo1b, which encodes a subunit of an NADPH oxidase evoking the oxidative stress. Furthermore, immunohistochemistry tests confirmed the structure-dependent attenuation of the overproduction of ROS and cardiac apoptosis. Our results verified the feasibility of bioisosteric replacement to attenuate the cardiotoxicity of 7-azaindazole-based PI3K inhibitors, suggesting that the screening for PI3K inhibitors with both high potency and low cardiotoxicity from bioisosteres would be a beneficial trial. [ABSTRACT FROM AUTHOR]

Background and Aim: Diabetes can causes disturbances in glucose homeostasis and negative changes in the heart muscle. Since physical activity in people with diabetes may affect the heart tissue, the aim of this study was to investigate the effect of high intensity interval training (HIIT) on content of FOXO3, PI3K and AKT proteins in the heart tissue of Wistar rats with type two diabetes. Materials and Methods: In the present study, 36 male Wistar rats were divided into four groups: diabetes - control, diabetes - exercise, control - healthy, and exercise - healthy. After two months of high-fat diet and induction of diabetes by Streptozotocin (35 mg/kg) in diabetes-control and diabetes-exercise groups, the animals in diabetes-exercise and exercise-healthy groups performed the HIIT protocol based on a percentage of Vmax achieved, that it was with two-minutes intervals and increasing number of intervals for eight weeks and five sessions per week. 48 hours after the last training session, cardiac tissue was extracted and the content of FOXO3, PI3K and AKT proteins were assessed using Western blotting. In addition, a histological study was performed at the tissue level using hematoxylin and Eosin staining. To analyze the data one-way analysis of variance and Tukey tests were used at a significance level of p<0.05. Results: The content of FOXO3 protein in diabetic groups significantly increased compared to healthy groups (p=0.001). In addition, the content of PI3K and AKT proteins in diabetes-control group also significantly decreased compared to healthy groups (p=0.001 and p=0.009, respectively), while the content of these two proteins significantly increased after training (p=0.01 and p=0.001, respectively). Moreover, at the tissue level of heart, the thickness and length of cardiac myocytes significantly increased due to diabetes (p=0.001); while after HIIT, this pathological hypertrophy reduced (p=0.02 and p=0.01, respectively). Conclusion: Finally, it can be stated that although the use of this training method did not show a change in the amount of FOXO3 protein, but it was able to increase the amount of PI3K and AKT proteins and improve the pathological hypertrophy caused by diabetes. [ABSTRACT FROM AUTHOR]

The primary symptom of diabetic encephalopathy (DE), a kind of central diabetic neuropathy caused by diabetes mellitus (DM), is cognitive impairment. In addition, the tetracyclic oxindole alkaloid isorhynchophylline (IRN) helps lessen cognitive impairment. However, it is still unclear how IRN affects DM and DE and what mechanisms are involved. The effectiveness of IRN on brain insulin resistance was carefully examined in this work, both in vitro and in vivo. We found that IRN accelerates spliced form of X-box binding protein 1 (sXBP1) translocation into the nucleus under high glucose conditions in vitro. IRN also facilitates the nuclear association of pCREB with sXBP1 and the binding of regulatory subunits of phosphatidylinositol 3-kinase (PI3K) p85α or p85β with XBP1 to restore high glucose impairment. Also, IRN treatment improves high glucose–mediated impairment of insulin signaling, endoplasmic reticulum stress, and pyroptosis/apoptosis by depending on sXBP1 in vitro. In vivo studies suggested that IRN attenuates cognitive impairment, ameliorating peripheral insulin resistance, activating insulin signaling, inactivating activating transcription factor 6 (ATF6) and C/EBP homology protein (CHOP), and mitigating pyroptosis/apoptosis by stimulation of sXBP1 nuclear translocation in the brain. In summary, these data indicate that IRN contributes to maintaining insulin homeostasis by activating sXBP1 in the brain. Thus, IRN is a potent antidiabetic agent as well as an sXBP1 activator that has promising potential for the prevention or treatment of DE. [ABSTRACT FROM AUTHOR]

Cue-elicited drug-craving is a cardinal feature of addiction that intensifies (incubates) during protracted withdrawal. In a rat model, these addiction-related behavioral pathologies are mediated, respectively, by time-dependent increases in PI3K/Akt1 signaling and reduced Group 1 metabotropic glutamate receptor (mGlu) expression, within the ventromedial prefrontal cortex (vmPFC). Herein, we examined the capacity of single oral dosing with everolimus, an FDA-approved inhibitor of the PI3K/Akt effector mTOR, to reduce incubated cocaine-craving and reverse incubation-associated changes in vmPFC kinase activity and mGlu expression. Rats were trained to lever-press for intravenous infusions of cocaine or delivery of sucrose pellets and then subjected to tests for cue-reinforced responding during early (3 days) or late (30-46 days) withdrawal. Rats were gavage-infused with everolimus (0-1.0 mg/kg), either prior to testing to examine for effects upon reinforcer-seeking behavior, or immediately following testing to probe effects upon the consolidation of extinction learning. Single oral dosing with everolimus dose-dependently blocked cocaine-seeking during late withdrawal and the effect lasted at least 24 h. No everolimus effects were observed for cue-elicited sucrose-seeking or cocaine-seeking in early withdrawal. In addition, everolimus treatment, following initial cue-testing, reduced subsequent cue hyper-responsivity exhibited observed during late withdrawal, arguing a facilitation of extinction memory consolidation. everolimus' "anti-incubation" effect was associated with a reversal of withdrawal-induced changes in indices of PI3K/Akt1/mTOR activity, as well as Homer protein and mGlu1/5 expression, within the prelimbic (PL) subregion of the prefrontal cortex. Our results indicate mTOR inhibition as a viable strategy for interrupting heightened cocaine-craving and facilitating addiction recovery during protracted withdrawal.

PurposeSomatic activating variants in the PI3K-AKT pathway cause vascular malformations with and without overgrowth. We previously reported an individual with capillary and lymphatic malformation harboring a pathogenic somatic variant in PIK3R1, which encodes three PI3K complex regulatory subunits. Here, we investigate PIK3R1 in a large cohort with vascular anomalies and identify an additional 16 individuals with somatic mosaic variants in PIK3R1.MethodsAffected tissue from individuals with vascular lesions and overgrowth recruited from a multisite collaborative network was studied. Next-generation sequencing targeting coding regions of cell-signaling and cancer-associated genes was performed followed by assessment of variant pathogenicity.ResultsThe phenotypic and variant spectrum associated with somatic variation in PIK3R1 is reported herein. Variants occurred in the inter-SH2 or N-terminal SH2 domains of all three PIK3R1 protein products. Phenotypic features overlapped those of the PIK3CA-related overgrowth spectrum (PROS). These overlapping features included mixed vascular malformations, sandal toe gap deformity with macrodactyly, lymphatic malformations, venous ectasias, and overgrowth of soft tissue or bone.ConclusionSomatic PIK3R1 variants sharing attributes with cancer-associated variants cause complex vascular malformations and overgrowth. The PIK3R1-associated phenotypic spectrum overlaps with PROS. These data extend understanding of the diverse phenotypic spectrum attributable to genetic variation in the PI3K-AKT pathway.

With the increasing prevalence of type 2 diabetes and fatty liver disease, there is still an unmet need to better treat hyperglycemia and hyperlipidemia. Here, we identify isthmin-1 (Ism1) as an adipokine and one that has a dual role in increasing adipose glucose uptake while suppressing hepatic lipid synthesis. Ism1 ablation results in impaired glucose tolerance, reduced adipose glucose uptake, and reduced insulin sensitivity, demonstrating an endogenous function for Ism1 in glucose regulation. Mechanistically, Ism1 activates a PI3K-AKT signaling pathway independently of the insulin and insulin-like growth factor receptors. Notably, while the glucoregulatory function is shared with insulin, Ism1 counteracts lipid accumulation in the liver by switching hepatocytes from a lipogenic to a protein synthesis state. Furthermore, therapeutic dosing of recombinant Ism1 improves diabetes in diet-induced obese mice and ameliorates hepatic steatosis in a diet-induced fatty liver mouse model. These findings uncover an unexpected, bioactive protein hormone that might have simultaneous therapeutic potential for diabetes and fatty liver disease.

Background and purposeHalofuginone is a febrifugine derivative originally isolated from Chinese traditional herb Chang Shan that exhibits anti-hypertrophic, anti-fibrotic and anti-proliferative effects. We sought to investigate whether halofuginone induced pulmonary vasodilation and attenuates chronic hypoxia-induced pulmonary hypertension (HPH).Experimental approachPatch-clamp experiments were conducted to examine the activity of voltage-dependent Ca2+ channels (VDCCs) in pulmonary artery smooth muscle cells (PASMCs). Digital fluorescence microscopy was used to measure intracellular Ca2+ concentration in PASMCs. Isolated perfused and ventilated mouse lungs were used to measure pulmonary artery pressure (PAP). Mice exposed to hypoxia (10% O2 ) for 4 weeks were used as model of HPH for in vivo experiments.Key resultsHalofuginone increased voltage-gated K+ (Kv ) currents in PASMCs and K+ currents through KCNA5 channels in HEK cells transfected with KCNA5 gene. HF (0.03-1 μM) inhibited receptor-operated Ca2+ entry in HEK cells transfected with calcium-sensing receptor gene and attenuated store-operated Ca2+ entry in PASMCs. Acute (3-5 min) intrapulmonary application of halofuginone significantly and reversibly inhibited alveolar hypoxia-induced pulmonary vasoconstriction dose-dependently (0.1-10 μM). Intraperitoneal administration of halofuginone (0.3 mg·kg-1 , for 2 weeks) partly reversed established PH in mice.Conclusion and implicationsHalofuginone is a potent pulmonary vasodilator by activating Kv channels and blocking VDCC and receptor-operated and store-operated Ca2+ channels in PASMCs. The therapeutic effect of halofuginone on experimental PH is probably due to combination of its vasodilator effects, via inhibition of excitation-contraction coupling and anti-proliferative effects, via inhibition of the PI3K/Akt/mTOR signalling pathway.

Objective To investigate the effect of melatonin on pancreatic autophagy in rats with gestational diabetes mellitus (GDM) and the underlying mechanism. Methods Twenty-eight rats were randomly divided into A group (rats with normal pregnancy), B group (rats with GDM), C group (low-dose melatonin administration for rats with GDM), and D group (high-dose melatonin administration for rats with GDM), with seven rats in each group. All the groups were examined for the se-rum levels of glucose, insulin, and glycosylated hemoglobin. Pancreatic tissue slices were prepared to examine the pathological changes of the pancreas with hematoxylin-eosin (HE) staining, the apoptosis of pancreatic cells with TUNEL staining, and the autophagy of the pancreatic tissue with transmission electron microscopy. Western blot was used to measure the expression of autop-hagy-related proteins in the pancreatic tissue: microtubule-associated protein 1 light chain 3 (LC3), Beclin1, phosphorylated phosphatidylinositol 3-kinase (p-PI3K), PI3K, phosphorylated protein kinase B (p-AKT), AKT, phosphorylated mammalian target of rapamycin (p-mTOR), and mTOR. Results There were significant differences in serum glucose, insulin, and glycosylated hemoglobin levels between different groups (F=52.26-66.73,P

During the early phase of tumorigenesis, primary malignant cells survive within a low nutrition environment caused by a poorly organized vascular system. Here, we sought to determine the functional significance of CD133 in the survival of cancer cells under nutrient-poor conditions. Knockdown and overexpression experiments demonstrated that CD133 suppresses colon cancer cell death induced by serum deprivation through activation of Akt-mediated anti-apoptosis and protein synthesis pathways. Furthermore, serum deprivation increased the amount of endogenous CD133 protein, which was regulated at least in part by phosphoinositide 3-kinase. Thus, it is highly likely that CD133 contributes to the acquisition/maintenance of the resistance to stress arising from nutrient deficiency in early avascular tumor tissues.

OBJECTIVE: We sought to identify novel genes and to establish the contribution of known genes in a large cohort of patients with nonsyndromic sporadic polymicrogyria and epilepsy. METHODS: We enrolled participants with polymicrogyria and their parents through the Epilepsy Phenome/Genome Project. We performed phenotyping and whole exome sequencing (WES), trio analysis, and gene-level collapsing analysis to identify de novo or inherited variants, including germline or mosaic (postzygotic) single nucleotide variants, small insertion-deletion (indel) variants, and copy number variants present in leukocyte-derived DNA. RESULTS: Across the cohort of 86 individuals with polymicrogyria and epilepsy, we identified seven with pathogenic or likely pathogenic variants in PIK3R2, including four germline and three mosaic variants. PIK3R2 was the only gene harboring more than expected de novo variants across the entire cohort, and likewise the only gene that passed the genome-wide threshold of significance in the gene-level rare variant collapsing analysis. Consistent with previous reports, the PIK3R2 phenotype consisted of bilateral polymicrogyria concentrated in the perisylvian region with macrocephaly. Beyond PIK3R2, we also identified one case each with likely causal de novo variants in CCND2 and DYNC1H1 and biallelic variants in WDR62, all genes previously associated with polymicrogyria. Candidate genetic explanations in this cohort included single nucleotide de novo variants in other epilepsy-associated and neurodevelopmental disease-associated genes (SCN2A in two individuals, GRIA3, CACNA1C) and a 597-kb deletion at 15q25, a neurodevelopmental disease susceptibility locus. SIGNIFICANCE: This study confirms germline and postzygotically acquired de novo variants in PIK3R2 as an important cause of bilateral perisylvian polymicrogyria, notably with macrocephaly. In total, trio-based WES identified a genetic diagnosis in 12% and a candidate diagnosis in 6% of our polymicrogyria cohort. Our results suggest possible roles for SCN2A, GRIA3, CACNA1C, and 15q25 deletion in polymicrogyria, each already associated with epilepsy or other neurodevelopmental conditions without brain malformations. The role of these genes in polymicrogyria will be further understood as more patients with polymicrogyria undergo genetic evaluation.

Targeted therapies for gastrointestinal stromal tumor (GIST) are modestly effective, but GIST cannot be cured with single agent tyrosine kinase inhibitors. In this study, we sought to identify new therapeutic targets in GIST by investigating the tumor microenvironment. Here, we identified a paracrine signaling network by which cancer-associated fibroblasts (CAFs) drive GIST growth and metastasis. Specifically, CAFs isolated from human tumors were found to produce high levels of platelet-derived growth factor C (PDGFC), which activated PDGFC-PDGFRA signal transduction in GIST cells that regulated the expression of SLUG, an epithelial-mesenchymal transition (EMT) transcription factor and downstream target of PDGFRA signaling. Together, this paracrine induce signal transduction cascade promoted tumor growth and metastasis in vivo. Moreover, in metastatic GIST patients, SLUG expression positively correlated with tumor size and mitotic index. Given that CAF paracrine signaling modulated GIST biology, we directly targeted CAFs with a dual PI3K/mTOR inhibitor, which synergized with imatinib to increase tumor cell killing and in vivo disease response. Taken together, we identified a previously unappreciated cellular target for GIST therapy in order to improve disease control and cure rates.

The role of Pi3K inhibitors in lymphoma is diminishing due to the adverse results from trials in indolent lymphoma, but is a one‐size‐fits‐all approach to drug development penalising some lymphoma subtypes and the newer generation of Pi3K inhibitors? The report by Soumerai et al. of zandelisib with zanubrutinib in follicular and mantle cell lymphoma is an important addition to the data. Commentary on: Soumerai et al. Safety and efficacy of zandelisib plus zanubrutinib in previously treated follicular and mantle cell lymphomas. Br J Haematol 2024;204:1762‐1770. [ABSTRACT FROM AUTHOR]

In glucose-starved cells, macroautophagy (hereafter referred to as autophagy) is considered to serve as an energy-generating process contributing to cell survival. AMPK (adenosine monophosphate-activated protein kinase) is the primary cellular energy sensor that is activated during glucose starvation. According to the current paradigm in the field, AMPK promotes autophagy in response to energy deprivation by binding and phosphorylating ULK1 (UNC−51 like kinase 1), the protein kinase responsible for autophagy initiation. However, conflicting findings have been reported casting doubts about the current established model. In our recent study, we have thoroughly reevaluated the role of AMPK in autophagy. Contrary to the current paradigm, our study revealed that AMPK functions as a negative regulator of ULK1 activity. The study has elucidated the underlying mechanism and demonstrated the significance of the negative role in controlling autophagy and maintaining cellular resilience during energy depletion. Abbreviations: AMPK: adenosine monophosphate-activated protein kinase; ULK1: UNC−51 like kinase 1; MTORC1: mechanistic target of rapamycin complex 1; ATG14: autophagy-related protein 14; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; ATP: adenosine triphosphate; VPS34: vacuolar protein sorting 34; BECN1: Beclin 1; AMPKα: AMPK catalytic subunit α; LKB1: liver kinase B1; PIK3R4: phosphatidylinositol 3-kinase regulatory subunit 4. [ABSTRACT FROM AUTHOR]

Myelodysplastic syndrome (MDS) is a clonal malignancy that develops from hematopoietic stem cells (HSCs), but the underlying mechanisms of MDS initiation are not well understood. The phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway is often dysregulated in MDS. To investigate how PI3K inactivation affects HSC function, we generated a mouse model in which three Class IA PI3K genes were deleted in hematopoietic cells. Surprisingly, PI3K deficiency caused cytopenias, reduced survival, and multilineage dysplasia with chromosomal abnormalities, consistent with MDS initiation. PI3K-deficient HSCs had impaired autophagy, and pharmacologic treatment with autophagy-inducing reagents improved HSC differentiation. Furthermore, a similar autophagic degradation defect was observed in MDS patient HSCs. Therefore, our study uncovered a crucial protective role for Class IA PI3K in maintaining autophagic flux in HSCs to preserve the balance between self-renewal and differentiation. [ABSTRACT FROM AUTHOR]

In the prodromal phase of neurodegenerative diseases, microglia switch to an activated state resulting in increased secretion of pro-inflammatory factors. We reported that C – C chemokine ligand 3 (CCL3), C – C chemokine ligand 4 (CCL4) and C – C chemokine ligand 5 (CCL5) contained in the secretome of activated microglia inhibit neuronal autophagy via a non-cell autonomous mechanism. These chemokines bind and activate neuronal C – C chemokine receptor type 5 (CCR5), which, in turn, promotes phosphoinositide 3-kinase (PI3K) – protein kinase B (PKB, or AKT) – mammalian target of rapamycin complex 1 (mTORC1) pathway activation, which inhibits autophagy, thus causing the accumulation of aggregate-prone proteins in the cytoplasm of neurons. The levels of CCR5 and its chemokine ligands are increased in the brains of pre-manifesting Huntington disease (HD) and tauopathy mouse models. CCR5 accumulation might be due to a self-amplifying mechanism, since CCR5 is a substrate of autophagy and CCL5-CCR5-mediated autophagy inhibition impairs CCR5 degradation. Furthermore, pharmacological, or genetic inhibition of CCR5 rescues mTORC1-autophagy dysfunction and improves neurodegeneration in HD and tauopathy mouse models, suggesting that CCR5 hyperactivation is a pathogenic signal driving the progression of these diseases. [ABSTRACT FROM AUTHOR]

The recruitment of ATG12–ATG5-ATG16L1 complex to phagophore mediated by the specific interaction between ATG16L1 and WIPI2, is pivotal to the formation of autophagosomes during macroautophagy. Recently, we reported that ATG16L1 contains two distinct WIPI2-binding sites, the previously reported WIPI2-binding site (WBS1), and the newly identified site (WBS2). By determining the crystal structures of WIPI2 with ATG16L1 WBS1 and WBS2 respectively, we uncovered that, unlike ATG16L1 WBS1, ATG16L1 WBS2 and its binding mechanism to WIPI2 are conserved from yeast to mammals. Using cell-based functional assays, we further demonstrated that the integrity of two WIPI2-binding sites of ATG16L1 is essential for normal autophagic flux. In summary, our study provided mechanistic insights into the interaction of two key autophagic proteins, ATG16L1 and WIPI2, and revealed a dual-binding-site mode adopted by ATG16L1 to associate with WIPI2. Abbreviations: ATG: autophagy-related protein; CCD: coiled-coil domain; ITC: isothermal titration calorimetry; PI3KC3-C1: class III phosphatidylinositol 3-kinase complex I; PtdIns3P: phosphatidylinositol-3-phosphate; ULK: Unc-51-like kinase; WBS: WIPI2-binding site; WIPI: WD repeat domain phosphoinositide-interacting protein. [ABSTRACT FROM AUTHOR]

BACKGROUNDAlthough CDK4/6 inhibitors are an established treatment for hormone receptor-positive, HER2-negative metastatic breast cancers, their benefit in other malignancies remains limited.METHODSWe investigated factors associated with clinical outcomes from CDK4/6 inhibitor-based therapy among patients with G1/S phase cell-cycle alterations (CDK4/6 amplifications, CCND1/2/3 amplifications, or CDKN2A/B alterations).RESULTSOverall, 2457 patients with diverse solid tumors that underwent clinical-grade, next-generation sequencing (182-465 genes) and therapy outcome of (non-breast cancer) patients treated with matched CDK4/6 inhibitors were analyzed. G1/S phase cell-cycle alterations occurred in 20.6% (507 of 2457) of patients; 99% of those patients (n = 501) harbored ≥1 characterized co-alteration (median, 4; range, 0-24). In 40 patients with G1/S phase cell-cycle alterations given CDK4/6 inhibitors as part of their regimen, significantly longer median progression-free survival (PFS) was observed when CDK4/6 inhibitor-based therapies matched a larger proportion of tumor alterations, often because CDK4/6 inhibitors were administered together with other drugs that were matched to genomic co-alterations, hence achieving a high matching score (high vs. low [≥50% vs.

The molecular mechanisms of cellular insulin action have been the focus of much investigation since the discovery of the hormone 100 years ago. Insulin action is impaired in metabolic syndrome, a condition known as insulin resistance. The actions of the hormone are initiated by binding to its receptor on the surface of target cells. The receptor is an α2β2 heterodimer that binds to insulin with high affinity, resulting in the activation of its tyrosine kinase activity. Once activated, the receptor can phosphorylate a number of intracellular substrates that initiate discrete signaling pathways. The tyrosine phosphorylation of some substrates activates phosphatidylinositol-3-kinase (PI3K), which produces polyphosphoinositides that interact with protein kinases, leading to activation of the kinase Akt. Phosphorylation of Shc leads to activation of the Ras/MAP kinase pathway. Phosphorylation of SH2B2 and of Cbl initiates activation of G proteins such as TC10. Activation of Akt and other protein kinases produces phosphorylation of a variety of substrates, including transcription factors, GTPase-activating proteins, and other kinases that control key metabolic events. Among the cellular processes controlled by insulin are vesicle trafficking, activities of metabolic enzymes, transcriptional factors, and degradation of insulin itself. Together these complex processes are coordinated to ensure glucose homeostasis.

Optogenetics has been harnessed to shed new mechanistic light on current and future therapeutic strategies. This has been to date achieved by the regulation of ion flow and electrical signals in neuronal cells and neural circuits that are known to be affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and are implicated in degenerative disorders, has never been demonstrated in an animal model of disease. Here, we reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1B9 flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinson's disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-кB pathway. Our results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to inspire novel strategies towards a spatio-temporal regulation of tissue repair.

Oxylipins modulate the behavior of immune cells in inflammation. Soluble epoxide hydrolase (sEH) converts anti-inflammatory epoxyeicosatrienoic acid (EET) to dihydroxyeicosatrienoic acid (DHET). An sEH-inhibitor, TPPU, has been demonstrated to ameliorate lipopolysaccharide (LPS)- and sepsis-induced inflammation via EETs. The immunomodulatory role of DHET is not well characterized. We hypothesized that TPPU dampens inflammation and that sEH-derived DHET alters neutrophil functionality in burn induced inflammation. Outbred mice were treated with vehicle, TPPU or 14,15-DHET and immediately subjected to either sham or dorsal scald 28% total body surface area burn injury. After 6 and 24 h, interleukin 6 (IL-6) serum levels and neutrophil activation were analyzed. For in vitro analyses, bone marrow derived neutrophil functionality and mRNA expression were examined. In vivo, 14,15-DHET and IL-6 serum concentrations were decreased after burn injury with TPPU administration. In vitro, 14,15-DHET impaired neutrophil chemotaxis, acidification, CXCR1/CXCR2 expression and reactive oxygen species (ROS) production, the latter independent from p38MAPK and PI3K signaling. We conclude that TPPU administration decreases DHET post-burn. Furthermore, DHET downregulates key neutrophil immune functions and mRNA expression. Altogether, these data reveal that TPPU not only increases anti-inflammatory and inflammation resolving EET levels, but also prevents potential impairment of neutrophils by DHET in trauma.

Hepatoblastoma (HB) is the predominant primary liver tumor in children. While the prognosis is favorable when the tumor can be resected, the outcome is dismal for patients with progressed HB. Therefore, a better understanding of the molecular mechanisms responsible for HB is imperative for early detection and effective treatment. Sequencing analysis of human HB specimens unraveled the pivotal role of Wnt/β-catenin pathway activation in this disease. Nonetheless, β-catenin activation alone does not suffice to induce HB, implying the need for additional alterations. Perturbations of several pathways, including Hippo, Hedgehog, NRF2/KEAP1, HGF/c-Met, NK-1R/SP, and PI3K/AKT/mTOR cascades and aberrant activation of c-MYC, n-MYC, and EZH2 proto-oncogenes, have been identified in HB, although their role requires additional investigation. Here, we summarize the current knowledge on HB molecular pathogenesis, the relevance of the preclinical findings for the human disease, and the innovative therapeutic strategies that could be beneficial for the treatment of HB patients.

The phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway regulates proliferation, survival and metabolism, and its dysregulation is one of the most frequent oncogenic events across human malignancies. Over the last two decades, there has been significant focus on the clinical development of PI3K pathway inhibitors. More than 40 different inhibitors of this axis have reached various stages of clinical trials, but only a few of them have been approved by the Food and Drug Administration (FDA) for cancer treatment. These clinical results, however, could be improved given the importance of PI3K signaling in cancer and its role in linking cancer growth with metabolism. In this systematic review, after a glance at PI3K/AKT/mTOR pathway and its different inhibitors, we retrieved registered clinical trials evaluating the efficacy and safety of PI3K/AKT/mTOR inhibitors on Clinicaltrials.gov. Following the extraction of the data, finally we analyzed 2250 included studies in multiple steps, beginning with an overview and moving on to the details about type of malignancies, inhibitors, and treatment strategies. We also took a closer look at more than 100 phase III-IV clinical trials to pinpoint promising therapies, hoping that presenting a comprehensive picture of current clinical trials casts a flash of light on what remains to be done in future clinical trials of PI3K/AKT/mTOR inhibitors in human malignancies. [ABSTRACT FROM AUTHOR]

BACKGROUND: Autophagy can play an important role in liver fibrosis by inducing the progression of hepatic stellate cell activation. Previous studies have shown that Rougan Jiangmei Formula could protect liver function, anti-inflammation, and anti-oxidative stress response. The mechanism of fighting liver fibrosis through autophagy has not been elucidated. OBJECTIVE: To investigate the effect of Rougan Jiangmei Formula medicated serum on activation and autophagy of human hepatic stellate cells induced by lipopolysaccharide and its underlying mechanism. METHODS: Human hepatic stellate cell activation models were established using lipopolysaccharide and divided into various treatment groups: (1) Blank group: human hepatic stellate cells+blank serum; (2) model group: human hepatic stellate cells+blank serum+lipopolysaccharide; (3) low-, medium- and high-dose Rougan Jiangmei Formula medicated serum groups: human hepatic stellate cells+low-, medium- and high-dose Rougan Jiangmei Formula medicated serum+lipopolysaccharide; (4) high-dose Rougan Jiangmei Formula medicated serum combined with rapamycin group: based on high-dose group+rapamycin; (5) high-dose Rougan Jiangmei Formula medicated serum combined with PI3K inhibitor group: based on high-dose group+LY294002. Cell proliferation was observed by CCK-8 assay. The autophagy was observed by monodansylcadaverine staining. The effects of Rougan Jiangmei Formula medicated serum on the expression levels of cell pathway-related proteins (PI3K, P-PI3K, AKT, P-AKT, mTOR, P-mTOR), autophagy-related proteins (P62, Beclin-1, LC3-I, LC3-II), the activation marker protein of hepatic stellate cells (α-smooth-muscle actin and collagen I) were determined by western blot assay. RESULTS AND CONCLUSION: (1) Rougan Jiangmei Formula medicated serum significantly reduced the proliferation of activated human hepatic stellate cells, down-regulated the protein expression of α-smooth-muscle actin and collagen I, up-regulated the expression of autophagy protein P62, and decreased the expression of Beclin-1 and LC3-II in a dose-dependent manner. (2) Monodansylcadaverine staining results exhibited that Rougan Jiangmei Formula medicated serum significantly inhibited the fluorescence intensity of the cell green spots. (3) After the addition of autophagy agonist rapamycin, the expression of autophagy protein P62 was reduced and the expression levels of Beclin-1 and LC3-II were increased. (4) After Rougan Jiangmei Formula medicated serum treatment, the phosphorylation levels of PI3K, Akt, and mTOR proteins were increased in a dose-dependent manner. This effect could be inhibited by the PI3K inhibitor LY294002. (5) It is indicated that Rougan Jiangmei Formula medicated serum may inhibit autophagy via the PI3K/Akt/mTOR signaling pathway and then reduce lipopolysaccharide-induced human hepatic stellate cell activation. [ABSTRACT FROM AUTHOR]

Background: Lung cancer remains the leading cause of cancer-related death worldwide. Targeted therapies with tyrosine kinase inhibitors (TKIs) result in improvement in survival for non-small cell lung cancer (NSCLC) with activating mutations of the epidermal growth factor receptor (EGFR). Unfortunately, most patients who initially respond to EGFR-TKI ultimately develop resistance to therapy, resulting in cancer progression and relapse. Combination therapy is today a common strategy for the treatment of tumors to increase the success rate, improve the outcome and survival of patients, and avoid the selection of resistant cancer cells through the activation of compensatory pathways. In NSCLC, the phosphoinositide-3-kinase/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway has been heavily implicated in both tumorigenesis and the progression of disease. Objectives: In this study, we investigated the efficacy of a PI3K δ-sparing inhibitor, MEN1611, in models of NSCLC sensitive and resistant to EGFR inhibitors (erlotinib and gefitinib) with a wild-type PIK3CA gene. Methods: We performed functional, biochemical, and immunohistochemistry studies. Results: We demonstrated good efficacy of MEN1611 in NSCLC devoid of PIK3CA gene mutations but with constitutive activation of the PI3K/AKT pathway and its synergistic effect with gefitinib both in vitro and in vivo. Conclusions: Overall, this preclinical study indicates that the inhibitor could be a candidate for the treatment of NSCLC with an erlotinib/gefitinib-resistant phenotype and constitutive activation of the PI3K/AKT pathway, a phenotype mimicked by our model system. [ABSTRACT FROM AUTHOR]

KRAS is the most commonly mutated oncogene. Targeted therapies have been developed against mediators of key downstream signaling pathways, predominantly components of the RAF/MEK/ERK kinase cascade. Unfortunately, single-agent efficacy of these agents is limited both by intrinsic and acquired resistance. Survival of drug-tolerant persister cells within the heterogeneous tumor population and/or acquired mutations that reactivate receptor tyrosine kinase (RTK)/RAS signaling can lead to outgrowth of tumor-initiating cells (TICs) and drive therapeutic resistance. Here, we show that targeting the key RTK/RAS pathway signaling intermediates SOS1 (Son of Sevenless 1) or KSR1 (Kinase Suppressor of RAS 1) both enhances the efficacy of, and prevents resistance to, the MEK inhibitor trametinib in KRAS-mutated lung (LUAD) and colorectal (COAD) adenocarcinoma cell lines depending on the specific mutational landscape. The SOS1 inhibitor BI-3406 enhanced the efficacy of trametinib and prevented trametinib resistance by targeting spheroid-initiating cells in KRASG12/G13-mutated LUAD and COAD cell lines that lacked PIK3CA comutations. Cell lines with KRASQ61 and/or PIK3CA mutations were insensitive to trametinib and BI-3406 combination therapy. In contrast, deletion of the RAF/MEK/ERK scaffold protein KSR1 prevented drug-induced SIC upregulation and restored trametinib sensitivity across all tested KRAS mutant cell lines in both PIK3CA-mutated and PIK3CA wild-type cancers. Our findings demonstrate that vertical inhibition of RTK/RAS signaling is an effective strategy to prevent therapeutic resistance in KRAS-mutated cancers, but therapeutic efficacy is dependent on both the specific KRAS mutant and underlying comutations. Thus, selection of optimal therapeutic combinations in KRAS-mutated cancers will require a detailed understanding of functional dependencies imposed by allele-specific KRAS mutations. [ABSTRACT FROM AUTHOR]

The kidney flushes out toxic substances and metabolic waste products, and homeostasis is maintained owing to the kidney efforts. Unfortunately, kidney disease is one of the illnesses with a poor prognosis and a high death rate. The current investigation was set out to assess erythropoietin (EPO) potential therapeutic benefits against thioacetamide (TAA)-induced kidney injury in rats. EPO treatment improved kidney functions, ameliorated serum urea, creatinine, and malondialdehyde, increased renal levels of reduced glutathione, and slowed the rise of JAK2, STAT5, AMPK, and their phosphorylated forms induced by TAA. EPO treatment also greatly suppressed JAK2, Phosphatidylinositol 3-kinases, and The Protein Kinase R-like ER Kinase gene expressions and mitigated the histopathological alterations brought on by TAA toxicity. EPO antioxidant and anti-inflammatory properties protected TAA-damaged kidneys. EPO regulates AMPK, JAK2/STAT5, and pro-inflammatory mediator synthesis. [ABSTRACT FROM AUTHOR]

Cell-to-cell variability during TNFα stimulated Tumor Necrosis Factor Receptor 1 (TNFR1) signaling can lead to single-cell level pro-survival and apoptotic responses. This variability stems from the heterogeneity in signal flow through intracellular signaling entities that regulate the balance between these two phenotypes. Using systematic Boolean dynamic modeling of a TNFR1 signaling network, we demonstrate that the signal flow path variability can be modulated to enable cells favour apoptosis. We developed a computationally efficient approach "Boolean Modeling based Prediction of Steady-state probability of Phenotype Reachability (BM-ProSPR)" to accurately predict the network's ability to settle into different phenotypes. Model analysis juxtaposed with the experimental observations revealed that NFκB and PI3K transient responses guide the XIAP behaviour to coordinate the crucial dynamic cross-talk between the pro-survival and apoptotic arms at the single-cell level. Model predicted the experimental observations that ~31% apoptosis increase can be achieved by arresting Comp1 – IKK* activity which regulates the NFκB and PI3K dynamics. Arresting Comp1 – IKK* activity causes signal flow path re-wiring towards apoptosis without significantly compromising NFκB levels, which govern adequate cell survival. Priming an ensemble of cancerous cells with inhibitors targeting the specific interaction involving Comp1 and IKK* prior to TNFα exposure could enable driving them towards apoptosis. [ABSTRACT FROM AUTHOR]

Coordinated cell movement is a fundamental process in organ formation. During heart development, bilateral myocardial precursors collectively move toward the midline (cardiac fusion) to form the primitive heart tube. Extrinsic influences such as the adjacent anterior endoderm are known to be required for cardiac fusion. We previously showed however, that the platelet-derived growth factor receptor alpha (Pdgfra) is also required for cardiac fusion (Bloomekatz et al., 2017). Nevertheless, an intrinsic mechanism that regulates myocardial movement has not been elucidated. Here, we show that the phosphoinositide 3-kinase (PI3K) intracellular signaling pathway has an essential intrinsic role in the myocardium directing movement toward the midline. In vivo imaging further reveals midline-oriented dynamic myocardial membrane protrusions that become unpolarized in PI3K-inhibited zebrafish embryos where myocardial movements are misdirected and slower. Moreover, we find that PI3K activity is dependent on and interacts with Pdgfra to regulate myocardial movement. Together our findings reveal an intrinsic myocardial steering mechanism that responds to extrinsic cues during the initiation of cardiac development. [ABSTRACT FROM AUTHOR]