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.

Prussian blue analogues-derived zero valent iron to efficiently activate peroxymonosulfate for phenol degradation triggered via reactive oxygen species and high-valent iron-oxo complexes.

It is of great significance to develop the effective technique to treat phenol-containing wastewater. Herein, Fe-based prussian blue analogues-derived zero valent iron (ZVI) was successfully synthesized by one-step calcination method. Owing to high specific surface area and rich active sites, ZVI-2 possessed excellent performance in charge transfer. Notably, in comparison with conventional ZVI and Fe2+, ZVI-2 can effectively activate peroxymonosulfate (PMS) for achieving rapid degradation of phenol, and the highest removal efficiency of phenol reached 94.9% within 24 min. More importantly, developed ZVI-2/PMS oxidation system with good stability displayed strong anti-interference capability. Interestingly, Fe0 loaded on the surface of ZVI-2 can efficiently break the O-O bond of PMS to generate reactive oxygen species (i.e., SO4•-, OH•, O2•- and 1O2). As main adsorption sites of PMS, the existence of oxygen vacancy promote the formation of high-valent transition metal complexes (namely ZVI-2≡Fe4+=O). Under the combined action of reactive oxygen species and ZVI-2≡Fe4+=O, phenol can be eventually degraded into CO2 and H2O. The possible degradation pathways of phenol were also investigated. Furthermore, proposed ZVI-2/PMS oxidation system displayed great potential for application in the field of wastewater treatment. All in all, current work provided a valuable reference for design and application of Fe-based catalysts in PS-AOPs.

Precise editing of FGFR3-TACC3 fusion genes with CRISPR-Cas13a in glioblastoma.

FGFR3-TACC3 (F3-T3) gene fusions are regarded as a "low-hanging fruit" paradigm for precision therapy in human glioblastoma (GBM). Small molecules designed to target the kinase in FGFR currently serve as one form of potential treatment but cause off-target effects and toxicity. Here, CRISPR-Cas13a, which is known to directly suppress gene expression at the transcriptional level and induce a collateral effect in eukaryotes, was leveraged as a possible precision therapy in cancer cells harboring F3-T3 fusion genes. A library consisting of crRNAs targeting the junction site of F3-T3 was designed, and an in silico simulation scheme was created to select the optimal crRNA candidates. An optimal crRNA, crRNA1, showed efficiency and specificity in inducing the collateral effect in only U87 cells expressing F3-T3 (U87-F3-T3). Expression profiles obtained with microarray analysis were consistent with induction of the collateral effect by the CRISPR-Cas13a system. Tumor cell proliferation and colony formation were decreased in U87-F3-T3 cells expressing the Cas13a-based tool, and tumor growth was suppressed in an orthotopic tumor model in mice. These findings demonstrate that the CRISPR-Cas13a system induces the collateral damage effect in cancer cells and provides a viable strategy for precision tumor therapy based on the customized design of a CRISPR-Cas13a-based tool against F3-T3 fusion genes.

Urinary metabolomic changes and microbiotic alterations in presenilin1/2 conditional double knockout mice.

BACKGROUND: Given the clinical low efficient treatment based on mono-brain-target design in Alzheimer's disease (AD) and an increasing emphasis on microbiome-gut-brain axis which was considered as a crucial pathway to affect the progress of AD along with metabolic changes, integrative metabolomic signatures and microbiotic community profilings were applied on the early age (2-month) and mature age (6-month) of presenilin1/2 conditional double knockout (PS cDKO) mice which exhibit a series of AD-like phenotypes, comparing with gender and age-matched C57BL/6 wild-type (WT) mice to clarify the relationship between microbiota and metabolomic changes during the disease progression of AD. MATERIALS AND METHODS: Urinary and fecal samples from PS cDKO mice and gender-matched C57BL/6 wild-type (WT) mice both at age of 2 and 6 months were collected. Urinary metabolomic signatures were measured by the gas chromatography-time-of-flight mass spectrometer, as well as 16S rRNA sequence analysis was performed to analyse the microbiota composition at both ages. Furthermore, combining microbiotic functional prediction and Spearman's correlation coefficient analysis to explore the relationship between differential urinary metabolites and gut microbiota. RESULTS: In addition to memory impairment, PS cDKO mice displayed metabolic and microbiotic changes at both of early and mature ages. By longitudinal study, xylitol and glycine were reduced at both ages. The disturbed metabolic pathways were involved in glycine, serine and threonine metabolism, glyoxylate and dicarboxylate metabolism, pentose and glucuronate interconversions, starch and sucrose metabolism, and citrate cycle, which were consistent with functional metabolic pathway predicted by the gut microbiome, including energy metabolism, lipid metabolism, glycan biosynthesis and metabolism. Besides reduced richness and evenness in gut microbiome, PS cDKO mice displayed increases in Lactobacillus, while decreases in norank_f_Muribaculaceae, Lachnospiraceae_NK4A136_group, Mucispirillum, and Odoribacter. Those altered microbiota were exceedingly associated with the levels of differential metabolites. CONCLUSIONS: The urinary metabolomics of AD may be partially mediated by the gut microbiota. The integrated analysis between gut microbes and host metabolism may provide a reference for the pathogenesis of AD.

Combination LSD1 and HOTAIR-EZH2 inhibition disrupts cell cycle processes and induces apoptosis in glioblastoma cells.

Glioblastoma (GBM) is the most common primary central nervous system tumor and has a poor prognosis, with a median survival time of only 14 months from diagnosis. Abnormally expressed long noncoding RNAs (lncRNAs) are important epigenetic regulators of chromatin modification and gene expression regulation in tumors, including GBM. We previously showed that the lncRNA HOTAIR is related to the cell cycle progression and can be used as an independent predictor in GBM. Lysine-specific demethylase 1 (LSD1), binding to 3' domain of HOTAIR, specifically removes mono- and di-methyl marks from H3 lysine 4 (H3K4) and plays key roles during carcinogenesis. In this study, we combined a HOTAIR-EZH2 disrupting agent and an LSD1 inhibitor, AC1Q3QWB (AQB) and GSK-LSD1, respectively, to block the two functional domains of HOTAIR and potentially provide therapeutic benefit in the treatment of GBM. Using an Agilent Human ceRNA Microarray, we identified tumor suppressor genes upregulated by AQB and GSK-LSD1, followed by Chromatin immunoprecipitation (ChIP) assays to explore the epigenetic mechanisms of genes activation. Microarray analysis showed that AQB and GSK-LSD1 regulate cell cycle processes and induces apoptosis in GBM cell lines. Furthermore, we found that the combination of AQB and GSK-LSD1 showed a powerful effect of inhibiting cell cycle processes by targeting CDKN1A, whereas apoptosis promoting effects of combination therapy were mediated by BBC3 in vitro. ChIP assays revealed that GSK-LSD1 and AQB regulate P21 and PUMA, respectively via upregulating H3K4me2 and downregulating H3K27me3. Combination therapy with AQB and GSK-LSD1 on tumor malignancy in vitro and GBM patient-derived xenograft (PDX) models shows enhanced anti-tumor efficacy and appears to be a promising new strategy for GBM treatment through its effects on epigenetic regulation.

Identification of long non-coding RNA HERC2P2 as a tumor suppressor in glioma.

Long non-coding RNAs (lncRNAs) have been reported to play important roles in glioma; however, most of them promote glioma progression. We constructed a competing endogenous (ceRNA) network based on the Chinese Glioma Genome Atlas dataset, and lncRNA hect domain and RLD 2 pseudogene 2 (HERC2P2) is the core of this network. Highly connected genes in the ceRNA network classified the glioma patients into three clusters with significantly different survival rates. The expression of HERC2P2 is positively correlated with survival and negatively correlated with clinical grade. Cell colony formation, Transwell and cell scratch tests were performed to evaluate the role of HERC2P2 in glioblastoma growth. Furthermore, we overexpressed HERC2P2 in U87 cells and established a mouse intracranial glioma model to examine the function of HERC2P2 in vivo. In conclusion, we identified a lncRNA with tumor suppressor functions in glioma that could be a potential biomarker for glioma patients.

Regulation of Hepatic Cholesteryl Ester Transfer Protein Expression and Reverse Cholesterol Transport by Inhibition of DNA Topoisomerase II.

Cholesteryl ester transfer protein (CETP) transfers cholesteryl esters from high density lipoprotein to triglyceride-rich lipoproteins. CETP expression can be transcriptionally activated by liver X receptor (LXR). Etoposide and teniposide are DNA topoisomerase II (Topo II) inhibitors. Etoposide has been reported to inhibit atherosclerosis in rabbits with un-fully elucidated mechanisms. In this study we determined if Topo II activity can influence cholesterol metabolism by regulating hepatic CETP expression. Inhibition of Topo II by etoposide, teniposide, or Topo II siRNA increased CETP expression in human hepatic cell line, HepG2 cells, which was associated with increased CETP secretion and mRNA expression. Meanwhile, inhibition of LXR expression by LXR siRNA attenuated induction of CETP expression by etoposide and teniposide. Etoposide and teniposide induced LXRα expression and LXRα/ß nuclear translocation while inhibiting expression of receptor interacting protein 140 (RIP140), an LXR co-repressor. In vivo, administration of teniposide moderately reduced serum lipid profiles, induced CETP expression in the liver, and activated reverse cholesterol transport in CETP transgenic mice. Our study demonstrates a novel function of Topo II inhibitors in cholesterol metabolism by activating hepatic CETP expression and reverse cholesterol transport.

Tamoxifen induces the development of hernia in mice by activating MMP-2 and MMP-13 expression.

Hernia is a disease with defects in collagen synthesis/metabolism. However, the underlying mechanisms for hernia formation have not been fully defined. Tamoxifen is a selective estrogen receptor modulator and used for patients with breast cancer. Tamoxifen also has pleiotropic and side effects. Herein, we report that tamoxifen treatment resulted in an appearance of a large bulge in the low abdomen between the hind legs in male but not in female mice. The autopsy demonstrated that the low abdominal wall was broken and a large amount of intestine herniated out of the abdominal cavity. Histological analysis indicated that tamoxifen caused structural abnormalities in the low abdominal wall which were associated with decreased type II collagen content. Furthermore, we determined increased matrix metalloproteinase-2 (MMP-2) and MMP-13 expression in the tissue. In vitro, tamoxifen induced MMP-2 and MMP-13 expression in fibroblasts. The promoter activity analysis and ChIP assay demonstrate that induction of MMP-13 expression was associated with activation of JNK-AP-1 and ERK1/2 signaling pathways while induction of MMP-2 expression was related to activation of the ERK1/2 signaling pathway. Taken together, our study establishes a novel murine hernia model, defines a severe side effect of tamoxifen, and suggests a caution to male patients receiving tamoxifen treatment.

Inhibition of ERK1/2 and activation of LXR synergistically reduce atherosclerotic lesions in ApoE-deficient mice.

OBJECTIVE: Activation of liver X receptor (LXR) inhibits atherosclerosis but induces hypertriglyceridemia. In vitro, it has been shown that mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor synergizes LXR ligand-induced macrophage ABCA1 expression and cholesterol efflux. In this study, we determined whether MEK1/2 (U0126) and LXR ligand (T0901317) can have a synergistic effect on the reduction of atherosclerosis while eliminating LXR ligand-induced fatty livers and hypertriglyceridemia. We also set out to identify the cellular mechanisms of the actions. APPROACH AND RESULTS: Wild-type mice were used to determine the effect of U0126 on a high-fat diet or high-fat diet plus T0901317-induced transient dyslipidemia and liver injury. ApoE deficient (apoE(-/-)) mice or mice with advanced lesions were used to determine the effect of the combination of T0901317 and U0126 on atherosclerosis and hypertriglyceridemia. We found that U0126 protected animals against T0901317-induced transient or long-term hepatic lipid accumulation, liver injury, and hypertriglyceridemia. Meanwhile, the combination of T0901317 and U0126 inhibited the development of atherosclerosis in a synergistic manner and reduced advanced lesions. Mechanistically, in addition to synergistic induction of macrophage ABCA1 expression, the combination of U0126 and T0901317 maintained arterial wall integrity, inhibited macrophage accumulation in aortas and formation of macrophages/foam cells, and activated reverse cholesterol transport. The inhibition of T0901317-induced lipid accumulation by the combined U0126 might be attributed to inactivation of lipogenesis and activation of lipolysis/fatty acid oxidation pathways. CONCLUSIONS: Our study suggests that the combination of mitogen-activated protein kinase kinase 1/2 inhibitor and LXR ligand can function as a novel therapy to synergistically reduce atherosclerosis while eliminating LXR-induced deleterious effects.

Inhibition of tumor growth by U0126 is associated with induction of interferon-γ production.

Several MEK1/2 inhibitors have been in clinical trial evaluation for cancer treatment. Interferon-γ (IFN-γ) is a cytokine with multiple biological functions including antitumor activity. Expression of IFN-γ can be induced by liver X receptor (LXR), a ligand-activated transcription factor. However, it remains unknown if the anti-cancer action of MEK1/2 inhibitors is completed, at least in part, by activating IFN-γ expression. In this study, we determined that U0126, a MEK1/2 inhibitor, increased tumor-free and survival rates and decreased growth of inoculated Lewis lung carcinomas in wild type mice. However, the protective effects were substantially attenuated in IFN-γ deficient (IFN-γ-/-) mice. At cellular and molecular levels, MEK1/2 inhibitors increased IFN-γ protein and mRNA expression and activated natural IFN-γ promoter but not the IFN-γ promoters with mutations of the LXR responsive elements (LXREs). MEK1/2 inhibitors also enhanced formation of the LXRE-nuclear protein complexes by inducing LXR expression and nuclear translocation. Similarly, MEK1/2 siRNA inhibited phosphorylation of ERK1/2 by MEK1/2 while activated IFN-γ expression. In contrast, inhibition of LXR expression by siRNA blocked MEK1/2 inhibitors-induced IFN-γ expression. U0126 also inhibited chemicals-induced pulmonary carcinomas, which was associated with increased IFN-γ expression in the lung. Taken together, our study suggests that MEK1/2 inhibitors induce IFN-γ production in an LXR-dependent manner and the induction of IFN-γ expression can partially contribute to the anti-tumorigenic properties of U0126.

Identification of interferon-γ as a new molecular target of liver X receptor.

LXR (liver X receptor) is a ligand-activated transcription factor and plays an important role in regulation of lipid homoeostasis and inflammation. Several studies indicate that LXR inhibits IFN-γ (interferon γ)-induced biological responses; however, the influence of LXR on IFN-γ expression has not been fully elucidated. In the present study, we investigated the effects of LXR activation on IFN-γ expression at different levels. At the molecular level, we surprisingly observed that LXR ligand (T0901317) induced macrophage and T-cell IFN-γ protein expression which was associated with increased mRNA and secreted protein levels in culture medium. In contrast, selective inhibition of LXRα and/or LXRß expression by siRNA reduced IFN-γ expression. Promoter analysis defined the multiple LXREs (LXR-responsive elements) in the proximal region of the IFN-γ promoter. EMSAs and ChIP indicated that LXR activation enhanced the binding of LXR protein to these LXREs. In vivo, T0901317 increased wild-type mouse serum IFN-γ levels and IFN-γ expression in the lung and lymph nodes. Functionally, we observed that administration of T0901317 to wild-type mice increased rates of survival and being tumour-free, and inhibited tumour growth when the animals were inoculated with LLC1 carcinoma. In contrast, these protective effects were substantially attenuated in IFN-γ-knockout (IFN-γ-/-) mice, suggesting that the induction of IFN-γ production plays a critical role in T0901317-inhibited tumour growth. Taken together, the results of the present study show that IFN-γ is another molecular target of LXR activation, and it suggests a new mechanism by which LXR inhibits tumour growth.

DNA topoisomerase II inhibitors induce macrophage ABCA1 expression and cholesterol efflux-an LXR-dependent mechanism.

ATP-binding cassette transporter A1 (ABCA1) facilitates cholesterol efflux and thereby inhibits lipid-laden macrophage/foam cell formation and atherosclerosis. ABCA1 expression is transcriptionally regulated by activation of liver X receptor (LXR). Both etoposide and teniposide are DNA topoisomerase II (Topo II) inhibitors and are chemotherapeutic medications used in the treatment of various cancers. Interestingly, etoposide inhibits atherosclerosis in rabbits by unclear mechanisms. Herein, we report the effects of etoposide and teniposide on macrophage ABCA1 expression and cholesterol efflux. Both etoposide and teniposide increased macrophage free cholesterol efflux. This increase was associated with increased ABCA1 mRNA and protein expression. Etoposide and teniposide also increased ABCA1 promoter activity in an LXR-dependent manner and formation of the LXRE-LXR/RXR complex indicating that transcriptional induction had occurred. Expression of ABCG1 and fatty acid synthase (FAS), another two LXR-targeted genes, was also induced by etoposide and teniposide. In vivo, administration of mice with either etoposide or teniposide induced macrophage ABCA1 expression and enhanced reverse cholesterol transport from macrophages to feces. Taken together, our study indicates that etoposide and teniposide increase macrophage ABCA1 expression and cholesterol efflux that may be attributed to the anti-atherogenic properties of etoposide. Our study also describes a new function for Topo II inhibitors in addition to their role in anti-tumorigenesis.

Tamoxifen inhibits macrophage FABP4 expression through the combined effects of the GR and PPARγ pathways.

Macrophage adipocyte fatty acid-binding protein (FABP4) plays an important role in foam cell formation and development of atherosclerosis. Tamoxifen inhibits this disease process. In the present study, we determined whether the anti-atherogenic property of tamoxifen was related to its inhibition of macrophage FABP4 expression. We initially observed that tamoxifen inhibited macrophage/foam cell formation, but the inhibition was attenuated when FABP4 expression was selectively inhibited by siRNA.We then observed that tamoxifen and 4-hydroxytamoxifen inhibited FABP4 protein expression in primary macrophages isolated from both the male and female wild-type mice, suggesting that the inhibition is sex-independent. Tamoxifen and 4-hydroxytamoxifen inhibited macrophage FABP4 protein expression induced either by activation of GR (glucocorticoid receptor) or PPARγ (peroxisome-proliferator-activated receptor γ). Associated with the decreased protein expression, Fabp4 mRNA expression and promoter activity were also inhibited by tamoxifen and 4-hydroxytamoxifen, indicating transcriptional regulation. Analysis of promoter activity and EMSA/ChIP assays indicated that tamoxifen and 4-hydroxytamoxifen activated the nGRE (negative glucocorticoid regulatory element), but inhibited the PPRE (PPARγ regulatory element) in the Fabp4 gene. In vivo, administration of tamoxifen to ApoE (apolipoprotein E)-deficient (apoE-/-) mice on a high-fat diet decreased FABP4 expression in macrophages and adipose tissues as well as circulating FABP4 levels. Tamoxifen also inhibited FABP4 protein expression by human blood monocyte-derived macrophages. Taken together, the results of the present study show that tamoxifen inhibited FABP4 expression through the combined effects of GR and PPARγ signalling pathways. Our findings suggest that the inhibition of macrophage FABP4 expression can be attributed to the antiatherogenic properties of tamoxifen.

Levetiracetam: A Potent Sword against Microglia Polarization in Gliomas.

Crosstalk between tumor cells and peritumoral cells contributes to immunosuppressive microenvironment formation in glioblastomas (GBM). A recent study revealed that glioma stem cells activated neuronal activity to promote microglial M2 polarization, leading to GBM progression, which could be pharmacologically blocked by levetiracetam, providing a practical strategy for GBM immunotherapy. See related article by Guo et al., p. 1160.

HIF1α/ATF3 partake in PGK1 K191/K192 succinylation by modulating P4HA1/succinate signaling in glioblastoma.

BACKGROUND: Hypoxia is a pathological hallmark in most cancers, including glioblastoma (GBM). Hypoxic signaling activation and post-translational modification (PTM) of oncogenic proteins are well-studied in cancers. Accumulating studies indicate glycolytic enzyme PGK1 plays a crucial role in tumorigenesis, yet the underlying mechanisms remain unknown. METHODS: We first used ChIP assays to uncover the crosstalk between HIF1α and ATF3 and their roles in P4HA1 regulation. Protein degradation analysis, LC-MS/MS, and in vitro succinate production assays were performed to examine the effect of protein succinylation on GBM pathology. Seahorse assay measured the effects of PGK1 succinylation at K191/K192 or its mutants on glucose metabolism. We utilized an in vivo intracranial mouse model for biochemical studies to elucidate the impact of ATF3 and P4HA1 on aerobic glycolysis and the tumor immune microenvironment. RESULTS: We demonstrated that HIF1α and ATF3 positively and negatively regulate the transcription of P4HA1, respectively, leading to an increased succinate production and increased activation of HIF1α signaling. P4HA1 expression elevated the succinate concentration, resulting in the enhanced succinylation of PGK1 at the K191 and K192 sites. Inhibition of proteasomal degradation of PGK1 by succinylation significantly increased aerobic glycolysis to generate lactate. Furthermore, ATF3 overexpression and P4HA1 knockdown reduced succinate and lactate levels in GBM cells, inhibiting immune responses and tumor growth. CONCLUSIONS: Together, our study demonstrates that HIF1α/ATF3 participated in P4HA1/succinate signaling, which is the major regulator of succinate biosynthesis and PGK1 succinylation at K191 and K192 sites in GBM. The P4HA1/succinate pathway might be a novel and promising target for aerobic glycolysis in GBM.

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.

RUNX1/NPM1/H3K4me3 complex contributes to extracellular matrix remodeling via enhancing FOSL2 transcriptional activation in glioblastoma.

Extracellular matrix (ECM) remodeling has been implicated in the tumor malignant progression and immune escape in glioblastoma (GBM). Runt-related transcription factor 1 (RUNX1) is a vital transcriptional factor for promoting tumorigenesis and invasion in mesenchymal subtype of GBM. But the correlation between RUNX1 and ECM genes expression and regulatory mechanism of RUNX1 on ECM genes expression remain poorly understood to date. In this study, by using integral analysis of chromatin immunoprecipitation-sequencing and RNA sequencing, we reported that RUNX1 positively regulated the expression of various ECM-related genes, including Fibronectin 1 (FN1), Collagen type IV alpha 1 chain (COL4A1), and Lumican (LUM), in GBM. Mechanistically, we demonstrated that RUNX1 interacted with Nucleophosmin 1 (NPM1) to maintain the chromatin accessibility and facilitate FOS Like 2, AP-1 Transcription Factor Subunit (FOSL2)-mediated transcriptional activation of ECM-related genes, which was independent of RUNX1's transcriptional function. ECM remodeling driven by RUNX1 promoted immunosuppressive microenvironment in GBM. In conclusion, this study provides a novel mechanism of RUNX1 binding to NPM1 in driving the ECM remodeling and GBM progression.

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.

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.