技术资料

The study introduces a new immunotherapy for treating melanoma and other cancers by developing a PROTAC that degrades NR4A1,an intracellular nuclear factor that plays a crucial role in immune suppression. An effective cancer therapy requires killing cancer cells and targeting the tumor microenvironment (TME). Searching for molecules critical for multiple cell types in the TME,we identified NR4A1 as one such molecule that can maintain the immune suppressive TME. Here,we establish NR4A1 as a valid target for cancer immunotherapy and describe a first-of-its-kind proteolysis-targeting chimera (PROTAC,named NR-V04) against NR4A1. NR-V04 degrades NR4A1 within hours in vitro and exhibits long-lasting NR4A1 degradation in tumors with an excellent safety profile. NR-V04 inhibits and frequently eradicates established tumors. At the mechanistic level,NR-V04 induces the tumor-infiltrating (TI) B cells and effector memory CD8+ T (Tem) cells and reduces monocytic myeloid-derived suppressor cells (m-MDSC),all of which are known to be clinically relevant immune cell populations in human melanomas. Overall,NR-V04–mediated NR4A1 degradation holds promise for enhancing anticancer immune responses and offers a new avenue for treating various types of cancers such as melanoma. Graphical Abstract View Publication

Loss-of-function mutations in the NF1 tumor suppressor result in deregulated Ras signaling and drive tumorigenesis in the familial cancer syndrome neurofibromatosis type I. However,the extent to which NF1 inactivation promotes sporadic tumorigenesis is unknown. Here we report that NF1 is inactivated in sporadic gliomas via two mechanisms: excessive proteasomal degradation and genetic loss. NF1 protein destabilization is triggered by the hyperactivation of protein kinase C (PKC) and confers sensitivity to PKC inhibitors. However,complete genetic loss,which only occurs when p53 is inactivated,mediates sensitivity to mTOR inhibitors. These studies reveal an expanding role for NF1 inactivation in sporadic gliomagenesis and illustrate how different mechanisms of inactivation are utilized in genetically distinct tumors,which consequently impacts therapeutic sensitivity. View Publication

H89 inhibits the dissociation-induced phosphorylation of PKA and two substrates of Rho-associated coiled-coil containing protein kinase (ROCK),myosin light chain (MLC2) and myosin phosphatase target subunit 1 (MYPT1),significantly increases cell survival and colony formation,and strongly depresses dissociation-induced cell death and cell blebbing without affecting the pluripotency of hESCs and their differentiation in vitro. View Publication

The tyrosine kinase Fyn plays a key role in oligodendrocyte differentiation and myelination in the central nervous system,but the molecules responsible for regulating Fyn activation in these processes remain poorly defined. Here we show that receptor-like protein-tyrosine phosphatase alpha (PTPalpha) is an important positive regulator of Fyn activation and signaling that is required for the differentiation of oligodendrocyte progenitor cells (OPCs). PTPalpha is expressed in OPCs and is up-regulated during differentiation. We used two model systems to investigate the role of PTPalpha in OPC differentiation: the rat CG4 cell line where PTPalpha expression was silenced by small interfering RNA,and oligosphere-derived primary OPCs isolated from wild-type and PTPalpha-null mouse embryos. In both cell systems,the ablation of PTPalpha inhibited differentiation and morphological changes that accompany this process. Although Fyn was activated upon induction of differentiation,the level of activation was severely reduced in cells lacking PTPalpha,as was the activation of Fyn effector molecules focal adhesion kinase,Rac1,and Cdc42,and inactivation of Rho. Interestingly,another downstream effector of Fyn,p190RhoGAP,which is responsible for Rho inactivation during differentiation,was not affected by PTPalpha ablation. In vivo studies revealed defective myelination in the PTPalpha(-/-) mouse brain. Together,our findings demonstrate that PTPalpha is a critical regulator of Fyn activation and of specific Fyn signaling events during differentiation,and is essential for promoting OPC differentiation and central nervous system myelination. View Publication

Serotonergic axons from the raphe nuclei in the brainstem project to every region of the brain,where they make connections through their extensive terminal arborizations. This serotonergic innervation contributes to various normal behaviors and psychiatric disorders. The protocadherin-alpha (Pcdha) family of clustered protocadherins consists of 14 cadherin-related molecules generated from a single gene cluster. We found that the Pcdhas were strongly expressed in the serotonergic neurons. To elucidate their roles,we examined serotonergic fibers in a mouse mutant (Pcdha(Delta CR/Delta CR)) lacking the Pcdha cytoplasmic region-encoding exons,which are common to the gene cluster. In the first week after birth,the distribution pattern of serotonergic fibers in Pcdha(Delta CR/Delta CR) mice was similar to wild-type,but by 3 weeks of age,when the serotonergic axonal termini complete their arborizations,the distribution of the projections was abnormal. In some target regions,notably the globus pallidus and substantia nigra,the normally even distribution of serotonin axonal terminals was,in the mutants,dense at the periphery of each region,but sparse in the center. In the stratum lacunosum-molecular of the hippocampus,the mutants showed denser serotonergic innervation than in wild-type,and in the dentate gyrus of the hippocampus and the caudate-putamen,the innervation was sparser. Together,the abnormalities suggested that Pcdha proteins are important in the late-stage maturation of serotonergic projections. Further examination of alternatively spliced exons encoding the cytoplasmic tail showed that the A-type (but not the B-type) cytoplasmic tail was essential for the normal development of serotonergic projections. View Publication

PTEN is a tumour suppressor frequently mutated in many types of cancers. Here we show that targeted disruption of PTEN leads to neoplastic transformation of human neural stem cells (NSCs),but not mesenchymal stem cells. PTEN-deficient NSCs display neoplasm-associated metabolic and gene expression profiles and generate intracranial tumours in immunodeficient mice. PTEN is localized to the nucleus in NSCs,binds to the PAX7 promoter through association with cAMP responsive element binding protein 1 (CREB)/CREB binding protein (CBP) and inhibits PAX7 transcription. PTEN deficiency leads to the upregulation of PAX7,which in turn promotes oncogenic transformation of NSCs and instates 'aggressiveness' in human glioblastoma stem cells. In a large clinical database,we find increased PAX7 levels in PTEN-deficient glioblastoma. Furthermore,we identify that mitomycin C selectively triggers apoptosis in NSCs with PTEN deficiency. Together,we uncover a potential mechanism of how PTEN safeguards NSCs,and establish a cellular platform to identify factors involved in NSC transformation,potentially permitting personalized treatment of glioblastoma. View Publication

Therapeutic modulation of phosphatidylinositol 3-kinase (PI3K)/PTEN signaling is currently being explored for multiple neurological indications including brain tumors and seizure disorders associated with cortical malformations. The effects of PI3K/PTEN signaling are highly cell context dependent but the function of this pathway in specific subsets of neural stem/progenitor cells generating oligodendroglial lineage cells has not been fully studied. To address this,we created Olig2-cre:Pten(fl/fl) mice that showed a unique pattern of Pten loss and PI3K activation in Olig2-lineage cells. Olig2-cre:Pten(fl/fl) animals progressively developed central nervous system white matter hypermyelination by 3 weeks of age leading to later onset leukodystrophy,chronic neurodegeneration,and death by 9 months. In contrast,during immediate postnatal development,oligodendroglia were unaffected but abnormal and accelerated differentiation of lateral subventricular zone stem cells produced calretinin-positive interneuron dysplasia. Neural stem cells isolated from Olig2-cre:Pten(fl/fl) mice also exhibited accelerated differentiation and proliferation into calretinin-positive interneurons and oligodendrocytes indicating such effects are cell autonomous. Opposition of the pathway by treatment of human primary neural progenitor cells (NPCs) with the PI3K inhibitor,NVP-BKM120,blocked in vitro differentiation of neurons and oligodendroglia indicating PI3K/PTEN effects on NPCs can be bidirectional. In summary,our results suggest Pten is a developmental rheostat regulating interneuron and oligodendroglial differentiation and support testing of PI3K modulating drugs as treatment for developmental and myelination disorders. However,such agents may need to be administered at ages that minimize potential effects on early stem/progenitor cell development. View Publication

The activity of neural precursor cells in the adult hippocampus is regulated by various stimuli; however,whether these stimuli regulate the same or different precursor populations remains unknown. Here,we developed a novel cell-sorting protocol that allows the purification to homogeneity of neurosphere-forming neural precursors from the adult mouse hippocampus and examined the responsiveness of individual precursors to various stimuli using a clonal assay. We show that within the Hes5-GFP(+)/Nestin-GFP(+)/EGFR(+) cell population,which comprises the majority of neurosphere-forming precursors,there are two distinct subpopulations of quiescent precursor cells,one directly activated by high-KCl depolarization,and the other activated by norepinephrine (NE). We then demonstrate that these two populations are differentially distributed along the septotemporal axis of the hippocampus,and show that the NE-responsive precursors are selectively regulated by GABA,whereas the KCl-responsive precursors are selectively modulated by corticosterone. Finally,based on RNAseq analysis by deep sequencing,we show that the progeny generated by activating NE-responsive versus KCl-responsive quiescent precursors are molecularly different. These results demonstrate that the adult hippocampus contains phenotypically similar but stimulus-specific populations of quiescent precursors,which may give rise to neural progeny with different functional capacity. View Publication

Thiamine deficiency (TD) leads to Wernicke's encephalopathy (WE),in which focal histological lesions occur in periventricular areas of the brain. Recently,impaired neurogenesis has been reported in the hippocampus during the dietary form of TD,and in pyrithiamine-induced TD (PTD),a well-characterized model of WE. To further characterize the consequences of PTD on neural stem/progenitor cell (NSPC) activity,we have examined the effect of this treatment in the rat on both the subventricular zone (SVZ) of the rostral lateral ventricle and subgranular layer (SGL) of the hippocampus,and in the thalamus and inferior colliculus,two vulnerable brain regions in this disorder. In both the SVZ and SGL,PTD led to a decrease in the numbers of bromodeoxyuridine-stained cells,indicating that proliferation of NSPCs destined for neurogenesis in these areas was reduced. Doublecortin (DCX) immunostaining in the SGL was decreased,indicating a reduction in neuroblast formation,consistent with impaired NSPC activity. DCX labeling was not apparent in focal areas of vulnerability. In the thalamus,proliferation of cells was absent while in the inferior colliculus,numerous actively dividing cells were apparent,indicative of a differential response between these two brain regions. Exposure of cultured neurospheres to PTD resulted in decreased proliferation of NSPCs,consistent with our in vivo findings. Together,these results indicate that PTD considerably affects cell proliferation and neurogenesis activity in both neurogenic areas and parts of the brain known to display structural and functional vulnerability,confirming and extending recent findings on the effects of TD on neurogenesis. Future use of NSPCs in vitro may allow a closer and more detailed examination of the mechanism(s) underlying inhibition of these cells during TD. View Publication

Stem cells,including cancer stem cells (CSCs),require niches to maintain stemness,yet it is unclear how CSCs maintain stemness in the suboptimal environment outside their niches during invasion. Postnatal co-deletion of Pten and Trp53 in mouse neural stem cells (NSCs) leads to the expansion of these cells in their subventricular zone (SVZ) niches but fails to maintain stemness outside the SVZ. We discovered that Qki is a major regulator of NSC stemness. Qk deletion on a Pten-/-; Trp53-/- background helps NSCs maintain their stemness outside the SVZ in Nes-CreERT2; QkL/L; PtenL/L; Trp53L/L mice,which develop glioblastoma with a penetrance of 92% and a median survival time of 105 d. Mechanistically,Qk deletion decreases endolysosome-mediated degradation and enriches receptors essential for maintaining self-renewal on the cytoplasmic membrane to cope with low ligand levels outside niches. Thus,downregulation of endolysosome levels by Qki loss helps glioma stem cells (GSCs) maintain their stemness in suboptimal environments outside their niches. View Publication

The processes involved in malignant gliomas damage were quantitatively evaluated by microscopy. The near-infrared fluorescent dye IR700 that is conjugated to an anti-CD133 antibody (IR700-CD133) specifically targets malignant gliomas (U87MG) and stem cells (BT142) and is endocytosed into the cells. The gliomas are then photodamaged by the release of reactive oxygen species (ROS) and the heat induced by illumination of IR700 by a red laser,and the motility of the vesicles within these cells is altered as a result of cellular damage. To investigate these changes in motility,we developed a new method that measures fluctuations in the intensity of phase-contrast images obtained from small areas within cells. The intensity fluctuation in U87MG cells gradually decreased as cell damage progressed,whereas the fluctuation in BT142 cells increased. The endocytosed IR700 dye was co-localized in acidic organelles such as endosomes and lysosomes. The pH in U87MG cells,as monitored by a pH indicator,was decreased and then gradually increased by the illumination of IR700,while the pH in BT142 cells increased monotonically. In these experiments,the processes of cell damage were quantitatively evaluated according to the motility of vesicles and changes in pH. View Publication