CRISPR-Cas9 library screening combined with an exosome-targeted delivery system addresses tumorigenesis/TMZ resistance in the mesenchymal subtype of glioblastoma.

Rationale: The large-scale genomic analysis classifies glioblastoma (GBM) into three major subtypes, including classical (CL), proneural (PN), and mesenchymal (MES) subtypes. Each of these subtypes exhibits a varying degree of sensitivity to the temozolomide (TMZ) treatment, while the prognosis corresponds to the molecular and genetic characteristics of the tumor cell type. Tumors with MES features are predominantly characterized by the NF1 deletion/alteration, leading to sustained activation of the RAS and PI3K-AKT signaling pathways in GBM and tend to acquire drug resistance, resulting in the worst prognosis compared to other subtypes (PN and CL). Here, we used the CRISPR/Cas9 library screening technique to detect TMZ-related gene targets that might play roles in acquiring drug resistance, using overexpressed KRAS-G12C mutant GBM cell lines. The study identified a key therapeutic strategy to address the chemoresistance against the MES subtype of GBM. Methods: The CRISPR-Cas9 library screening was used to discover genes associated with TMZ resistance in the U87-KRAS (U87-MG which is overexpressed KRAS-G12C mutant) cells. The patient-derived GBM primary cell line TBD0220 was used for experimental validations in vivo and in vitro. Chromatin isolation by RNA purification (ChIRP) and chromatin immunoprecipitation (ChIP) assays were used to elucidate the silencing mechanism of tumor suppressor genes in the MES-GBM subtype. The small-molecule inhibitor EPIC-0412 was obtained through high-throughput screening. Transmission electron microscopy (TEM) was used to characterize the exosomes (Exos) secreted by GBM cells after TMZ treatment. Blood-derived Exos-based targeted delivery of siRNA, TMZ, and EPIC-0412 was optimized to tailor personalized therapy in vivo. Results: Using the genome-wide CRISPR-Cas9 library screening, we found that the ERBIN gene could be epigenetically regulated in the U87-KRAS cells. ERBIN overexpression inhibited the RAS signaling and downstream proliferation and invasion effects of GBM tumor cells. EPIC-0412 treatment inhibited tumor proliferation and EMT progression by upregulating the ERBIN expression both in vitro and in vivo. Genome-wide CRISPR-Cas9 screening also identified RASGRP1(Ras guanine nucleotide-releasing protein 1) and VPS28(Vacuolar protein sorting-associated protein 28) genes as synthetically lethal in response to TMZ treatment in the U87-KRAS cells. We found that RASGRP1 activated the RAS-mediated DDR pathway by promoting the RAS-GTP transformation. VPS28 promoted the Exos secretion and decreased intracellular TMZ concentration in GBM cells. The targeted Exos delivery system encapsulating drugs and siRNAs together showed a powerful therapeutic effect against GBM in vivo. Conclusions: We demonstrate a new mechanism by which ERBIN is epigenetically silenced by the RAS signaling in the MES subtype of GBM. Restoration of the ERBIN expression with EPIC-0412 significantly inhibits the RAS signaling downstream. RASGRP1 and VPS28 genes are associated with the promotion of TMZ resistance through RAS-GDP to RAS-GTP transformation and TMZ efflux, as well. A quadruple combination therapy based on a targeted Exos delivery system demonstrated significantly reduced tumor burden in vivo. Therefore, our study provides new insights and therapeutic approaches for regulating tumor progression and TMZ resistance in the MES-GBM subtype.

EPIC-0628 abrogates HOTAIR/EZH2 interaction and enhances the temozolomide efficacy via promoting ATF3 expression and inhibiting DNA damage repair in glioblastoma.

The efficacy of temozolomide (TMZ) treatment in glioblastoma (GBM) is influenced by various mechanisms, mainly including the level of O6-methylguanine-DNA methyltransferase (MGMT) and the activity of DNA damage repair (DDR) pathways. In our previous study, we had proved that long non-coding RNA HOTAIR regulated the GBM progression and mediated DDR by interacting with EZH2, the catalytic subunit of PRC2. In this study, we developed a small-molecule inhibitor called EPIC-0628 that selectively disrupted the HOTAIR-EZH2 interaction and promoted ATF3 expression. The upregulation of ATF3 inhibited the recruitment of p300, p-p65, p-Stat3 and SP1 to the MGMT promoter. Hence, EPIC-0628 silenced MGMT expression. Besides, EPIC-0628 induced cell cycle arrest by increasing the expression of CDKN1A and impaired DNA double-strand break repair via suppressing the ATF3-p38-E2F1 pathway. Lastly, EPIC-0628 enhanced TMZ efficacy in GBM in vitro and vivo. Hence, this study provided evidence for the combination of epigenetic drugs EPIC-0628 with TMZ for GBM treatment through the above mechanisms.

EPIC-1042 as a potent PTRF/Cavin1-caveolin-1 interaction inhibitor to induce PARP1 autophagic degradation and suppress temozolomide efflux for glioblastoma.

BACKGROUND: Temozolomide (TMZ) treatment efficacy in glioblastoma is determined by various mechanisms such as TMZ efflux, autophagy, base excision repair (BER) pathway, and the level of O6-methylguanine-DNA methyltransferase (MGMT). Here, we reported a novel small-molecular inhibitor (SMI) EPIC-1042 (C20H28N6) with the potential to decrease TMZ efflux and promote PARP1 degradation via autolysosomes in the early stage. METHODS: EPIC-1042 was obtained from receptor-based virtual screening. Co-immunoprecipitation and pull-down assays were applied to verify the blocking effect of EPIC-1042. Western blotting, co-immunoprecipitation, and immunofluorescence were used to elucidate the underlying mechanisms of EPIC-1042. In vivo experiments were performed to verify the efficacy of EPIC-1042 in sensitizing glioblastoma cells to TMZ. RESULTS: EPIC-1042 physically interrupted the interaction of PTRF/Cavin1 and caveolin-1, leading to reduced secretion of small extracellular vesicles (sEVs) to decrease TMZ efflux. It also induced PARP1 autophagic degradation via increased p62 expression that more p62 bound to PARP1 and specially promoted PARP1 translocation into autolysosomes for degradation in the early stage. Moreover, EPIC-1042 inhibited autophagy flux at last. The application of EPIC-1042 enhanced TMZ efficacy in glioblastoma in vivo. CONCLUSION: EPIC-1042 reinforced the effect of TMZ by preventing TMZ efflux, inducing PARP1 degradation via autolysosomes to perturb the BER pathway and recruitment of MGMT, and inhibiting autophagy flux in the later stage. Therefore, this study provided a novel therapeutic strategy using the combination of TMZ with EPIC-1042 for glioblastoma treatment.

MUC1 promotes glioblastoma progression and TMZ resistance by stabilizing EGFRvIII.

Epidermal growth factor receptor variant III (EGFRvIII) is a mutant isoform of EGFR with a deletion of exons 2-7 making it insensitive to EGF stimulation and downstream signal constitutive activation. However, the mechanism underlying the stability of EGFRvIII remains unclear. Based on CRISPR-Cas9 library screening, we found that mucin1 (MUC1) is essential for EGFRvIII glioma cell survival and temozolomide (TMZ) resistance. We revealed that MUC1-C was upregulated in EGFRvIII-positive cells, where it enhanced the stability of EGFRvIII. Knockdown of MUC1-C increased the colocalization of EGFRvIII and lysosomes. Upregulation of MUC1 occurred in an NF-κB dependent manner, and inhibition of the NF-κB pathway could interrupt the EGFRvIII-MUC1 feedback loop by inhibiting MUC1-C. In a previous report, we identified AC1Q3QWB (AQB), a small molecule that could inhibit the phosphorylation of NF-κB. By screening the structural analogs of AQB, we obtained EPIC-1027, which could inhibit the NF-κB pathway more effectively. EPIC-1027 disrupted the EGFRvIII-MUC1-C positive feedback loop in vitro and in vivo, inhibited glioma progression, and promoted sensitization to TMZ. In conclusion, we revealed the pivotal role of MUC1-C in stabilizing EGFRvIII in glioblastoma (GBM) and identified a small molecule, EPIC-1027, with great potential in GBM treatment.

Retina as a window to cerebral dysfunction following studies with circRNA signature during neurodegeneration.

Ischemia-induced cerebral injury is a major cause of dementia or death worldwide. The pre-diagnosis is still challenging due to the retarded symptoms. The retina is regarded as the extension of cerebral tissue. Circular RNAs have emerged as the crucial regulators in gene regulatory network and disease progression. However, it is still unknown whether circRNAs can be used as the common regulators and diagnostic markers for cerebral neurodegeneration and retinal neurodegeneration. Methods: C57BL/6J mice were subjected to transient middle cerebral artery occlusion and circRNA microarray profiling was performed to identify neurodegeneration-related circRNAs. Quantitative reverse-transcription PCR (qRT-PCR) assays were performed to verify circRNA expression pattern. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed to determine the biologic modules and signaling pathway. TTC staining, Nissl's staining, and immunofluorescence staining assays were performed to investigate the role of circRNA in cerebral neurodegeneration and retinal neurodegeneration in vivo. MTT assay, Propidium iodide (PI)/Calcein-AM staining, and Rhodamine 123 assays were performed to investigate the role of circRNA in neuronal injury in vitro. Bioinformatics, RIP, and luciferase activity assays were performed to determine the regulatory mechanism of circRNA in neurodegeneration. Results: 217 differentially expressed circRNAs were identified between ischemic cerebral tissues and normal controls. Among them, cGLIS3 was shown as the common regulator of cerebral neurodegeneration and retinal neurodegeneration. cGLIS3 silencing alleviated ischemia-induced retinal neurodegeneration and MCAO-induced cerebral neurodegeneration in vivo. cGLIS3 silencing protected against OGD/R-induced RGC injury in vitro. The circulating levels of cGLIS3 were significantly increased in the patients with ischemic stroke compared to healthy subjects. cGLIS3 levels were also increased in the aqueous humor of the patients with retinal vein occlusion. cGLIS3 regulated neuronal cell injury by acting as miR-203 sponge and its level was controlled by EIF4A3. Conclusions: This study provides molecular evidence that the retina is window of the brain from circRNA perspective. cGLIS3 is a common regulator and diagnostic marker of cerebral neurodegeneration and retinal neurodegeneration.

Ameliorative effect of Magnesium Isoglycyrrhizinate on hepatic encephalopathy by Epirubicin.

BACKGROUND: The purpose of the present study was to evaluate the protective effect of Magnesium Isoglycyrrhizinate (MI) on Epirubicin (EPI)-induced hepatic encephalopathy (HE) and explore its underlying mechanism. METHODS: Mice were divided randomly into groups for treatments as follows: control group, EPI group (Model group), EPI + MI (25, 50 mg/kg) group. Morris water maze test were conducted to evaluate the spatial learning and memory ability. The serum and hippocampus levels of oxidative stress or inflammation were uncovered with the detection of superoxide dismutase (SOD), malondialdehyde (MDA), and pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α). RESULTS: As a result, treatment with MI effectively ameliorated the EPI-induced decline in the ability of spatial learning and memory. MI also significantly relieved the severity of oxidative stress or inflammation in serum and hippocampus, which was accompanied with regulating liver functional parameters. Western blot data demonstrated that administration of MI could regulate the redox-related expressions of Txnip, Trx, Nrf2, HO-1, p-IκB-α, p-NF-κB, Caspase-3, Caspase-9, Bax and Bcl-2 in EPI-stimulated hepatic encephalopathy (HE). And the potency of MI treatments on Nrf2, NF-κB expression was also confirmed with immunohistochemical analysis. CONCLUSIONS: Taken together, the protective effect of Magnesium Isoglycyrrhizinate on EPI-induced hepatic encephalopathy might be mediated via the Txnip/Nrf2/NF-κB signaling pathway.

Design, synthesis and biological evaluation of 4-aniline-thieno[2,3-d]pyrimidine derivatives as MNK1 inhibitors against renal cell carcinoma and nasopharyngeal carcinoma.

MAP Kinase Interacting Serine/Threonine Kinase 1 (MNK1) play important roles in the signaling transduction of MAPK pathways. It is significantly overexpressed in renal clear cell carcinoma and head-neck squamous cell carcinoma tissues in both mRNA and protein levels. Based on the crystallographic structure of MNK1 protein and binding modes analysis of known MNK inhibitors, we have designed and synthesized a series of 4-aniline-thieno[2,3-d]pyrimidine derivatives as potential MNK1 inhibitors. These synthetic compounds are tested in biochemical and cell proliferation assays, and six of them display potent inhibitory capacity against MNK1 kinase and cancer cell lines. Compound 12dj with strongest inhibitory capacity is transferred to molecular mechanism studies, and the results indicated that 12dj remarkably suppresses the phosphorylation of EIF4E, a substrate of MNK1. And the expression levels of MNK1, ERK1/2 and pERK1/2 are not affected by compound 12dj incubation in SUNE-1 and 786-O cells. In summary, our works suggested that these novel 4-aniline-thieno[2,3-d]pyrimidine based MNK1 inhibitors might be attractive lead compounds for targeted therapy of renal cell carcinoma and nasopharyngeal carcinoma.

Deletion of aquaporin-4 aggravates brain pathology after blocking of the meningeal lymphatic drainage.

The glymphatic pathway and meningeal lymphatic vessels are involved in clearance of metabolic macromolecules from the brain. However, the functional interaction between the two systems in the maintenance of brain homeostasis remains unclear. Here we reported that deletion of aquaporin-4 (AQP4), a functional regulator of glymphatic clearance, aggravated brain pathology of 3 month-old mice after blocking of the meningeal lymphatic drainage for 2 weeks via ligation of the deep cervical lymphatic nodes (LdcLNs). LdcLNs increased total and phosphorylated Tau protein levels in the hippocampus of both genotype mice, but increased hippocampal amyloid beta 1-40 and 1-42 levels only in AQP4 null mice, with up-regulation of beta-site amyloid precursor protein-cleaving enzyme 1 and down-regulation of insulin degrading enzyme. Consistently, LdcLNs caused microglial reactivity and activation of nod-like receptor protein-3 inflammasomes in the AQP4 null hippocampus. These mice also showed hippocampal neuronal apoptosis and declines in exploring and cognitive abilities. Deletion of AQP4, but not LdcLNs, increased brain water content. Together, these findings have revealed respective and interactive roles of the glymphatic system and the dural lymphatic system in maintaining amyloid beta, Tau proteins and water homeostasis in the brain, helping to understand the pathogenesis of neurological diseases associated with mis-accumulation of brain macromolecules.

Depressive- and anxiety-like phenotypes in young adult APPSwe/PS1dE9 transgenic mice with insensitivity to chronic mild stress.

Early Alzheimer's disease (AD) and depression share many symptoms, but the underlying mechanisms are not clear. Therefore, characterizing the shared and different biological changes between the two disorders will be helpful in making an early diagnosis and planning treatment. In the present study, 8-week-old APPSwe/PS1dE9 transgenic mice received chronic mild stress (CMS) for 8 weeks followed by a series of behavioral, biochemical and pathological analyses. APPSwe/PS1dE9 mice showed depressive- and anxiety-like behaviors, and reduced sociability, accompanied by high levels of soluble beta-amyloid, glial activation, neuroinflammation and brain derived neurotrophic factor signaling disturbance in the hippocampus. Notably, APPSwe/PS1dE9 mice exposure to CMS partially aggravated anxiety-like states rather than depressive-like responses and sociability deficits, with further elevated hippocampal interleukin-6 and tumor necrosis factor-α levels. These results demonstrated that young adult APPSwe/PS1dE9 have depressive- and anxiety-like phenotypes that were resistant to CMS compared to wild-type mice. This finding may help to understand the pathogenic mechanism of psychiatric symptoms associated with early AD.

Synthesis, antitumor activity and molecular mechanism of doxorubicin conjugated trimethyl-chitosan polymeric micelle loading Beclin1 siRNA for drug-resisted bladder cancer therapy.

Herein, we describe a convenient approach for the preparation of a polymeric micelle using doxorubicin (DOX) conjugated trimethyl-chitosan (TMC) with Beclin-1 siRNA (Si-Beclin1/DOX-TMC). This micelle displayed a potent capacity for autophagy inhibition and reversed drug-resistance to DOX in BIU-87/ADR cell lines. The Si-Beclin1/DOX-TMC micelle was highly cytotoxic to both drug-sensitive BIU-87 and drug-resistant BIU-87/ADR cells. Its capacity to reverse drug-resistance was dependent upon upregulation of autophagy levels in BIU-87/ADR cells. DOX was conjugated to TMC via a pH-sensitive Schiff base, which responded to the acidic lysosome microenvironment and resulted in the cytoplasmic release of DOX. The structure of DOX conjugation to the TMC polymeric micelle was characterized by NMR, GPC, TEM and DLS. DOX release profiles in different pH environment were determined by HPLC. Cellular uptake, changes to nuclei morphology and formation of autophagosomes were observed using a fluorescence microscope. Finally, in vivo antitumor activity of systemic Si-Beclin1/DOX-TMC micelle administration was evaluated in BIU-87/ADR xenograft models and Si-Beclin1/DOX-TMC micelles showed significantly suppressed tumor growth.