参考文献

Timothy syndrome (TS) is a severe,multisystem disorder characterized by autism,epilepsy,long-QT syndrome and other neuropsychiatric conditions 1 . TS type 1 (TS1) is caused by a gain-of-function variant in the alternatively spliced and developmentally enriched CACNA1C exon 8A,as opposed to its counterpart exon 8. We previously uncovered several phenotypes in neurons derived from patients with TS1,including delayed channel inactivation,prolonged depolarization-induced calcium rise,impaired interneuron migration,activity-dependent dendrite retraction and an unanticipated persistent expression of exon 8A 2 – 6 . We reasoned that switching CACNA1C exon utilization from 8A to 8 would represent a potential therapeutic strategy. Here we developed antisense oligonucleotides (ASOs) to effectively decrease the inclusion of exon 8A in human cells both in vitro and,following transplantation,in vivo. We discovered that the ASO-mediated switch from exon 8A to 8 robustly rescued defects in patient-derived cortical organoids and migration in forebrain assembloids. Leveraging a transplantation platform previously developed 7,we found that a single intrathecal ASO administration rescued calcium changes and in vivo dendrite retraction of patient neurons,suggesting that suppression of CACNA1C exon 8A expression is a potential treatment for TS1. Broadly,these experiments illustrate how a multilevel,in vivo and in vitro stem cell model-based approach can identify strategies to reverse disease-relevant neural pathophysiology. Subject terms: Autism spectrum disorders,Development of the nervous system View Publication

Sinefungin,a naturally occurring antifungal antibiotic nucleoside containing an ornithine residue,linked by a C-C bond to C-5' of adenosine,cures mice infected with Trypanosoma brucei brucei,T. congolense,or T. vivax; the effect of the drug is more pronounced towards T. congolense. Anti-trypanosomal activity of sinefungin could be the result of the inhibition of transmethylation reactions or of polyamine biosynthesis--or both--in parasites. View Publication

The MAPK pathway is one of the most important pathways for novel anticancer drug development. We performed high-throughput screening for compounds that induce expression of p15INK4b,and identified JTP-74057 (GSK1120212),which is being evaluated in ongoing phase I,II and III clinical trials. We characterized its antitumor activities in vitro and in vivo. JTP-74057 strongly inhibited MEK1/2 kinase activities,but did not inhibit another 98 kinase activities. Treatment by JTP-74057 resulted in growth inhibition accompanied with upregulation of p15INK4b and/or p27KIP1 in most of the colorectal cancer cell lines tested. Daily oral administration of JTP-74057 for 14 days suppressed tumor growth of HT-29 and COLO205 xenografts in nude mice. Notably,tumor regression was observed only in COLO205 xenografts,and COLO205 was much more sensitive to JTP-74057-induced apoptosis than HT-29 in vitro. Treatment with an Akt inhibitor enhanced the JTP-74057-induced apoptosis in HT-29 cells. Finally,JTP-74057 exhibited an additive or a synergistic effect in combination with the standard-of-care agents,5-fluorouracil,oxaliplatin or SN-38. JTP-74057,a highly specific and potent MEK1/2 inhibitor,exerts favorable antitumor activities in vitro and in vivo. Sensitivity to JTP-74057-induced apoptosis may be an important factor for the estimation of in vivo efficacy,and sensitivity was enhanced by an Akt inhibitor. These results suggest the usefulness of JTP-74057 in therapeutic applications for colorectal cancer patients. View Publication

In this study,we examined the effects of isoform-specific functional inhibitors of lysophosphatidic acid acyltransferase (LPAAT),which converts lysophosphatidic acid to phosphatidic acid,on multiple myeloma (MM) cell growth and survival. The LPAAT-beta inhibitors CT-32176,CT-32458,and CT-32615 induced textgreater95% growth inhibition (P textless 0.01) in MM.1S,U266,and RPMI8226 MM cell lines,as well as MM cells from patients (IC(50),50-200 nM). We further characterized this LPAAT-beta inhibitory effect using CT-32615,the most potent inhibitor of MM cell growth. CT-32615 triggered apoptosis in MM cells via caspase-8,caspase-3,caspase-7,and poly (ADP-ribose) polymerase cleavage. Neither interleukin 6 nor insulin-like growth factor I inhibited CT-32615-induced apoptosis. Dexamethasone and immunomodulatory derivatives of thalidomide (IMiDs),but not proteasome inhibitor PS-341,augmented MM cell apoptosis triggered by LPAAT-beta inhibitors. CT-32615-induced apoptosis was associated with phosphorylation of p53 and c-Jun NH(2)-terminal kinase (JNK); conversely,JNK inhibitor SP600125 and dominant-negative JNK inhibited CT-32615-induced apoptosis. Importantly,CT-32615 inhibited tumor necrosis factor-alpha-triggered nuclear factor-kappaB activation but did not affect either tumor necrosis factor-alpha-induced p38 mitogen-activated protein kinase phosphorylation or interleukin 6-triggered signal transducers and activators of transcription 3 phosphorylation. Finally,although binding of MM cells to bone marrow stromal cells augments MM cell growth and protects against dexamethasone-induced apoptosis,CT-32615 induced apoptosis even of adherent MM cells. Our data therefore demonstrate for the first time that inhibiting LPAAT-beta induces cytotoxicity in MM cells in the bone marrow milieu,providing the framework for clinical trials of these novel agents in MM. View Publication

This study evaluated the effects of a mammalian target of mTOR inhibitor everolimus alone or in combination with trastuzumab on stem cells from HER2-overexpressing primary breast cancer cells and the BT474 breast cancer cell line in vitro and in vivo. For the in vitro studies,we sorted ESA(+)CD44(+)CD24(-/low) cells as stem cells from primary breast cancer cells and BT474 cells using flow cytometry. The MTT assay was used to quantify the inhibitory effect of the drugs on total cells and stem cells specifically. Stem cell apoptosis,cell cycle distributions,and their tumorigenicity after treatment were investigated by flow cytometry or soft agar colony formation assays. For the in vivo studies,BALB/c mice were injected with BT474 stem cells,and the different treatments were administered. After necropsy,the expression of Ki67,CD31,AKT1,and phospho-AKT (Thr308) was analyzed by immunohistochemistry. For the in vitro studies,Treatment with everolimus resulted in stem cell growth inhibition in a dose-dependent manner. The combination of everolimus with trastuzumab was more effective at inhibiting cell growth (P textless 0.001) and tumorigenicity (P textless 0.001) compared with single-agent therapy. In addition,an increase in G1 cell cycle arrest and an increased population of cells in early apoptosis were seen in the combination treatment group compared with either of the single-agent groups (P textless 0.01). For the in vivo studies,everolimus plus trastuzumab therapy was much more effective at reducing tumor volume in mice compared with either single agent alone (P textless 0.05). Compared with everolimus alone,the combination of everolimus and trastuzumab reduced the expression of Ki67,AKT1,and phospho-AKT (Thr308) (P textless 0.05). We conclude that everolimus has effective inhibitory effects on HER2-overexpressing stem cells in vitro and vivo. Everolimus plus trastuzumab is a rational combination treatment that may be promising in human clinical trials. View Publication

BACKGROUND: Combining MEK inhibitors with other signalling pathway inhibitors or conventional cytotoxic drugs represents a promising new strategy against cancer. RDEA119/BAY 869766 is a highly potent and selective MEK1/2 inhibitor undergoing phase I human clinical trials. The effects of RDEA119/BAY 869766 as a single agent and in combination with rapamycin were studied in 3 early passage primary pancreatic cancer xenografts,OCIP19,21,and 23,grown orthotopically. METHODS: Anti-cancer effects were determined in separate groups following chronic drug exposure. Effects on cell cycle and downstream signalling were examined by flow cytometry and western blot,respectively. Plasma RDEA119 concentrations were measured to monitor the drug accumulation in vivo. RESULTS: RDEA119/BAY 869766 alone or in combination with rapamycin showed significant growth inhibition in all the 3 models,with a significant decrease in the percentage of cells in S-phase,accompanied by a large decrease in bromodeoxyuridine labelling and cell cycle arrest predominantly in G1. The S6 ribosomal protein was inhibited to a greater extent with combination treatment in all the three models. Blood plasma pharmacokinetic analyses indicated that RDEA119 levels achieved in vivo are similar to those that produce target inhibition and cell cycle arrest in vitro. CONCLUSIONS: Agents targeting the ERK and mTOR pathway have anticancer activity in primary xenografts,and these results support testing this combination in pancreatic cancer patients. View Publication

Neural stem cells have intact innate immune responses that protect them from virus infection and cell death. Yet,viruses can antagonize such responses to establish neuropathogenesis. Using a forebrain organoid model system at two developmental time points,we identified that neural stem cells,in particular radial glia,are basally primed to respond to virus infection by upregulating several antiviral interferon-stimulated genes. Infection of these organoids with a neuropathogenic Enterovirus-D68 strain,demonstrated the ability of this virus to impede immune activation by blocking interferon responses. Together,our data highlight immune gene signatures present in different types of neural stem cells and differential viral capacity to block neural-specific immune induction.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-024-03275-5. View Publication

Bats are natural reservoirs for several zoonotic viruses,potentially due to an enhanced capacity to control viral infection. However,the mechanisms of antiviral responses in bats are poorly defined. Here we established a Jamaican fruit bat (JFB) intestinal organoid model of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. JFB organoids were susceptible to SARS-CoV-2 infection,with increased viral RNA and subgenomic RNA detected in cell lysates and supernatants. Gene expression of type I interferons and inflammatory cytokines was induced in response to SARS-CoV-2 but not in response to TLR agonists. Interestingly,SARS-CoV-2 did not lead to cytopathic effects in JFB organoids but caused enhanced organoid growth. Proteomic analyses revealed an increase in inflammatory signaling,cell turnover,cell repair,and SARS-CoV-2 infection pathways. Collectively,our findings suggest that primary JFB intestinal epithelial cells can mount a successful antiviral interferon response and that SARS-CoV-2 infection in JFB cells induces protective regenerative pathways. View Publication

Cancer stem-like cells are thought to contribute to tumor recurrence. The anthrax toxin receptor 1 (ANTXR1) has been identified as a functional biomarker of normal stem cells and breast cancer stem-like cells. Primary stem cell-enriched basal cells (CD49f(+)/EpCAM(-)/Lin(-)) expressed higher levels of ANTXR1 compared with mature luminal cells. CD49f(+)/EpCAM(-),CD44(+)/EpCAM(-),CD44(+)/CD24(-),or ALDEFLUOR-positive subpopulations of breast cancer cells were enriched for ANTXR1 expression. CD44(+)/CD24(-)/ANTXR1(+) cells displayed enhanced self-renewal as measured by mammosphere assay compared with CD44(+)/CD24(-)/ANTXR1(-) cells. Activation of ANTXR1 by its natural ligand C5A,a fragment of collagen VI $$3,increased stem cell self-renewal in mammosphere assays and Wnt signaling including the expression of the Wnt receptor-lipoprotein receptor-related protein 6 (LRP6),phosphorylation of GSK3$$/$$,and elevated expression of Wnt target genes. RNAi-mediated silencing of ANTXR1 enhanced the expression of luminal-enriched genes but diminished Wnt signaling including reduced LRP6 and ZEB1 expression,self-renewal,invasion,tumorigenicity,and metastasis. ANTXR1 silencing also reduced the expression of HSPA1A,which is overexpressed in metastatic breast cancer stem cells. Analysis of public databases revealed ANTXR1 amplification in medullary breast carcinoma and overexpression in estrogen receptor-negative breast cancers with the worst outcome. Furthermore,ANTXR1 is among the 10% most overexpressed genes in breast cancer and is coexpressed with collagen VI. Thus,ANTXR1:C5A interactions bridge a network of collagen cleavage and remodeling in the tumor microenvironment,linking it to a stemness signaling network that drives metastatic progression. View Publication

Sustained,indolent immune injury of the vasculature of a heart transplant limits long-term graft and recipient survival. This injury is mitigated by a poorly characterized,maladaptive repair response. Vascular endothelial cells respond to proangiogenic cues in the embryo by differentiation to specialized phenotypes,associated with expression of apelin. In the adult,the role of developmental proangiogenic cues in repair of the established vasculature is largely unknown. We found that human and minor histocompatibility-mismatched donor mouse heart allografts with alloimmune-mediated vasculopathy upregulated expression of apelin in arteries and myocardial microvessels. In vivo,loss of donor heart expression of apelin facilitated graft immune cell infiltration,blunted vascular repair,and worsened occlusive vasculopathy in mice. In vitro,an apelin receptor agonist analog elicited endothelial nitric oxide synthase activation to promote endothelial monolayer wound repair and reduce immune cell adhesion. Thus,apelin acted as an autocrine growth cue to sustain vascular repair and mitigate the effects of immune injury. Treatment with an apelin receptor agonist after vasculopathy was established markedly reduced progression of arterial occlusion in mice. Together,these initial data identify proangiogenic apelin as a key mediator of coronary vascular repair and a pharmacotherapeutic target for immune-mediated injury of the coronary vasculature. View Publication