Mesenchymal stem cell-derived exosomal microRNA-367-3p alleviates experimental autoimmune encephalomyelitis via inhibition of microglial ferroptosis by targeting EZH2.

Multiple sclerosis (MS) is an autoimmune, inflammatory demyelinating disorder of the central nervous system. Accumulating evidence has underscored the therapeutic potential of bone marrow mesenchymal stem cells (BMSCs)-derived exosomes (BMSC-Exos) containing bioactive compounds in MS. Herein, the current study sought to characterize the mechanism of BMSC-Exos harboring miR-367-3p both in BV2 microglia by Erastin-induced ferroptosis and in experimental autoimmune encephalomyelitis (EAE), a typical animal model of MS. Exosomes were firstly isolated from BMSCs and identified for further use. BV2 microglia were co-cultured with miR-367-3p-containing BMSC-Exos, followed by an assessment of cell ferroptosis. Mechanistic exploration was furthered by the interaction of miR-367-3p and its downstream regulators. Lastly, BMSC-Exos harboring miR-367-3p were injected into EAE mice for in vivo validation. BMSC-Exos carrying miR-367-3p restrained microglial ferroptosis in vitro. Mechanistically, miR-367-3p could bind to Enhancer of zeste homolog 2 (EZH2) and restrain EZH2 expression, leading to the over-expression of solute carrier family 7 member 11 (SLC7A11). Meanwhile, over-expression of SLC7A11 resulted in Glutathione Peroxidase 4 (GPX4) activation and ferroptosis suppression. Ectopic expression of EZH2 in vitro negated the protective effects of BMSC-Exos. Furthermore, BMSC-Exos containing miR-367-3p relieved the severity of EAE by suppressing ferroptosis and restraining EZH2 expression in vivo. Collectively, our findings suggest that BMSC-Exos carrying miR-367-3p brings about a significant decline in microglia ferroptosis by repressing EZH2 and alleviating the severity of EAE in vivo, suggesting a possible role of miR-367-3p overexpression in the treatment strategy of EAE. AVAILABILITY OF DATA AND MATERIALS: The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Exosomal miR-23b-3p from bone mesenchymal stem cells alleviates experimental autoimmune encephalomyelitis by inhibiting microglial pyroptosis.

Multiple sclerosis (MS) is a chronic autoimmune disease that affects the central nervous system and is marked by inflammation and damage to the myelin sheath surrounding nerve fibers. Recent studies have highlighted the therapeutic value of exosomes (Exos) obtained from bone marrow mesenchymal stem cells (BMSCs) in MS treatment. These BMSC-Exos contain biologically active molecules that show promising results in preclinical evaluations. The aim of this study was to investigate the mechanism of BMSC-Exos containing miR-23b-3p in both LPS-stimulated BV2 microglia and in experimental autoimmune encephalomyelitis (EAE), an animal model for MS. Exos were isolated from BMSCs, and their effects were evaluated in vitro by co-culturing with BV2 microglia. The interaction between miR-23b-3p and its downstream targets was also explored. The efficacy of BMSC-Exos was further verified in vivo by injecting the Exos into EAE mice. The results showed that BMSC-Exos containing miR-23b-3p reduced microglial pyroptosis in vivo by specifically binding to and suppressing the expression of NEK7. In vivo, BMSC-Exos containing miR-23b-3p alleviated the severity of EAE by decreasing microglial inflammation and pyroptosis via the repression of NEK7. These findings provide new insights into the therapeutic potential of BMSC-Exos containing miR-23b-3p for MS.

Pterostilbene Protects the Optic Nerves and Retina in a Murine Model of Experimental Autoimmune Encephalomyelitis via Activation of SIRT1 Signaling.

Optic neuritis and retinal damage are common manifestations of multiple sclerosis (MS). Pterostilbene (PT) has been used to treat multiple diseases for its anti-inflammatory, anti-apoptosis and neuroprotective activities. This study aimed to investigate whether PT exerts a therapeutic effect on optic neuritis and retinal damage triggered by MS. Here, experimental autoimmune encephalomyelitis (EAE), an experimental model for MS, was induced in female C57BL/6 mice by immunizing with MOG35-55 peptide and treating with pertussis toxin. The mice were intraperitoneally injected with 20 mg/kg and 40 mg/kg PT once daily for 25 days at 24 h post immunization. We found that PT alleviated EAE severity and delayed EAE onset. Moreover, PT mitigated EAE-induced optic nerves and retinal inflammation, as indicated by the decreased Iba-1+ and GFAP+ cells and mRNA levels of interleukin-6, tumor necrosis factor-α and interleukin-1ß and the increased Iba-1+sirtuin 1 (SIRT1)+ and GFAP+SIRT1+ cells in the optic nerves and retina. PT also protected the optic nerves against demyelination and axonal loss and the retina against disorders in retinal morphology and apoptosis of retinal ganglion cells. High-dose PT had a more significant effect on protection of the optic nerves and retina in EAE than low-dose PT. In addition, PT activated SIRT1 signaling in the optic nerves and retina. Notably, EX-527, an inhibitor of SIRT1, reversed the effect of high-dose PT on the optic nerves and retina, indicating that PT exerted the protective effect via activating SIRT1 signaling. This study provides a potential candidate for treating MS.

NAD+ improved experimental autoimmune encephalomyelitis by regulating SIRT1 to inhibit PI3K/Akt/mTOR signaling pathway.

OBJECTIVE: To investigate the effect of NAD+ on thymus autophagy in experimental autoimmune encephalomyelitis (EAE) mice through SIRT1. METHODS: Bioinformatic analysis was used to identify hub genes. Forty female C57BL/6 mice were randomly divided into 4 groups: control, EAE, NAD+, and NAD+ +SIRT1 inhibitor (SIRT-IN-3) groups and SIRT1 group. The NAD+ group and SIRT1 inhibitor group were treated with NAD+ drug and fed for 4 weeks. The neurological function scores were evaluated weekly. The thymus tissues of wild-type mice were removed, ground and filtered into single-cell suspension. MOG 35-55 (1 µg/mL) was given to primary thymic epithelial cells (TECs) to induce EAE model in vitro. The expression of LC-3A/B was observed by immunofluorescence. The expressions or the activation/phosphorylation of associated proteins were detected by Western blot. RESULTS: Enrichment analysis showed PI3K-Akt-mTOR and autophagy pathway were main terms in EAE diseases, and the relationship between NAD+ and SIRT1. The activation of p-PI3K, p-Akt and p-mTOR were the highest in the EAE group consistent with decreased P62, Beclin1, LC-3A/B and SIRT1, and NAD+ reversed these results, furthermore SIRT1 inhibitor: SIRT-IN3 weakened the NAD+' effects in both in vivo and in vitro experiments. Immunofluorescence study in vivo and in vitro were accord with the results of western blot. CONCLUSIONS: NAD+ exerted a protective effect on EAE mice by inhibiting PI3K/Akt/mTOR signaling pathway through SIRT1 in TECs, and prevented EAE mice from sustained damage.

Interplay between the EMT transcription factors ZEB1 and ZEB2 regulates hematopoietic stem and progenitor cell differentiation and hematopoietic lineage fidelity.

The ZEB2 transcription factor has been demonstrated to play important roles in hematopoiesis and leukemic transformation. ZEB1 is a close family member of ZEB2 but has remained more enigmatic concerning its roles in hematopoiesis. Here, we show using conditional loss-of-function approaches and bone marrow (BM) reconstitution experiments that ZEB1 plays a cell-autonomous role in hematopoietic lineage differentiation, particularly as a positive regulator of monocyte development in addition to its previously reported important role in T-cell differentiation. Analysis of existing single-cell (sc) RNA sequencing (RNA-seq) data of early hematopoiesis has revealed distinctive expression differences between Zeb1 and Zeb2 in hematopoietic stem and progenitor cell (HSPC) differentiation, with Zeb2 being more highly and broadly expressed than Zeb1 except at a key transition point (short-term HSC [ST-HSC]➔MPP1), whereby Zeb1 appears to be the dominantly expressed family member. Inducible genetic inactivation of both Zeb1 and Zeb2 using a tamoxifen-inducible Cre-mediated approach leads to acute BM failure at this transition point with increased long-term and short-term hematopoietic stem cell numbers and an accompanying decrease in all hematopoietic lineage differentiation. Bioinformatics analysis of RNA-seq data has revealed that ZEB2 acts predominantly as a transcriptional repressor involved in restraining mature hematopoietic lineage gene expression programs from being expressed too early in HSPCs. ZEB1 appears to fine-tune this repressive role during hematopoiesis to ensure hematopoietic lineage fidelity. Analysis of Rosa26 locus-based transgenic models has revealed that Zeb1 as well as Zeb2 cDNA-based overexpression within the hematopoietic system can drive extramedullary hematopoiesis/splenomegaly and enhance monocyte development. Finally, inactivation of Zeb2 alone or Zeb1/2 together was found to enhance survival in secondary MLL-AF9 acute myeloid leukemia (AML) models attesting to the oncogenic role of ZEB1/2 in AML.

The EMT modulator SNAI1 contributes to AML pathogenesis via its interaction with LSD1.

Modulators of epithelial-to-mesenchymal transition (EMT) have recently emerged as novel players in the field of leukemia biology. The mechanisms by which EMT modulators contribute to leukemia pathogenesis, however, remain to be elucidated. Here we show that overexpression of SNAI1, a key modulator of EMT, is a pathologically relevant event in human acute myeloid leukemia (AML) that contributes to impaired differentiation, enhanced self-renewal, and proliferation of immature myeloid cells. We demonstrate that ectopic expression of Snai1 in hematopoietic cells predisposes mice to AML development. This effect is mediated by interaction with the histone demethylase KDM1A/LSD1. Our data shed new light on the role of SNAI1 in leukemia development and identify a novel mechanism of LSD1 corruption in cancer. This is particularly pertinent given the current interest surrounding the use of LSD1 inhibitors in the treatment of multiple different malignancies, including AML.

Necrostatin-1 ameliorates the pathogenesis of experimental autoimmune encephalomyelitis by suppressing apoptosis and necroptosis of oligodendrocyte precursor cells.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system characterized by neuronal demyelination. MS pathogenesis occurs via multiple mechanisms, and is mediated in part by oligodendrocyte apoptosis and a robust inflammatory response. In the present study, Necrostatin-1 (Nec-1), a specific inhibitor of the receptor-interacting protein 1 kinase domain, was revealed to effectively alleviate the severity and pathological damage associated with experimental autoimmune encephalomyelitis (EAE), a commonly used mouse model of MS. In addition, treatment with Nec-1 significantly decreased the number of lesions and inflammatory cell infiltrates in spinal cord tissues, as well as the production of associated pro-inflammatory cytokines, including tumor necrosis factor α (TNFα), interferon-γ and interleukin-1ß. Nec-1 also suppressed TNFα + zVAD-fmk-induced apoptosis and necroptosis in primary oligodendrocyte precursor cells. The present study revealed that Nec-1 effectively attenuated the progression of EAE by suppressing apoptosis and necroptosis in oligodendrocytes, and represents a potential novel therapeutic agent for the treatment of MS.

ZEB2 and LMO2 drive immature T-cell lymphoblastic leukemia via distinct oncogenic mechanisms.

ZEB1 and ZEB2 are structurally related E-box binding homeobox transcription factors that induce epithelial to mesenchymal transitions during development and disease. As such, they regulate cancer cell invasion, dissemination and metastasis of solid tumors. In addition, their expression is associated with the gain of cancer stem cell properties and resistance to therapy. Using conditional loss-of-function mice, we previously demonstrated that Zeb2 also plays pivotal roles in hematopoiesis, controlling important cell fate decisions, lineage commitment and fidelity. In addition, upon Zeb2 overexpression, mice spontaneously develop immature T-cell lymphoblastic leukemia. Here we show that pre-leukemic Zeb2-overexpressing thymocytes are characterized by a differentiation delay at beta-selection due to aberrant activation of the interleukin-7 receptor signaling pathway. Notably, and in contrast to Lmo2-overexpressing thymocytes, these pre-leukemic Zeb2-overexpressing T-cell progenitors display no acquired self-renewal properties. Finally, Zeb2 activation in more differentiated T-cell precursor cells can also drive malignant T-cell development, suggesting that the early T-cell differentiation delay is not essential for Zeb2-mediated leukemic transformation. Altogether, our data suggest that Zeb2 and Lmo2 drive malignant transformation of immature T-cell progenitors via distinct molecular mechanisms.

Modelling ponatinib resistance in tyrosine kinase inhibitor-naïve and dasatinib resistant BCR-ABL1+ cell lines.

TKI resistance remains a major impediment to successful treatment of CML. In this study, we investigated the emerging modes of ponatinib resistance in TKI-naïve and dasatinib resistant BCR-ABL1+ cell lines. To investigate potential resistance mechanisms, ponatinib resistance was generated in BCR-ABL1+ cell-lines by long-term exposure to increasing concentrations of ponatinib. Two cell lines with prior dasatinib resistance demonstrated BCR-ABL1 kinase domain (KD) mutation(s) upon exposure to ponatinib. In one of these cell lines the T315I mutation had emerged during dasatinib exposure. When further cultured with ponatinib, the T315I mutation level and BCR-ABL1 mRNA expression level were increased. In the other cell line, compound mutations G250E/E255K developed with ponatinib exposure. In contrast, the ponatinib resistant cell lines that had no prior exposure to other TKIs (TKI-naïve) did not develop BCR-ABL1 KD mutations. Rather, both of these cell lines demonstrated Bcr-Abl-independent resistance via Axl overexpression. Axl, a receptor tyrosine kinase, has previously been associated with imatinib and nilotinib resistance. Ponatinib sensitivity was restored following Axl inhibition or shRNA-mediated-knockdown of Axl, suggesting that Axl was the primary driver of resistance and a potential target for therapy in this setting.

Increased peroxisome proliferator-activated receptor γ activity reduces imatinib uptake and efficacy in chronic myeloid leukemia mononuclear cells.

Imatinib is actively transported by organic cation transporter-1 (OCT-1) influx transporter, and low OCT-1 activity in diagnostic chronic myeloid leukemia blood mononuclear cells is significantly associated with poor molecular response to imatinib. Herein we report that, in diagnostic chronic myeloid leukemia mononuclear cells and BCR-ABL1+ cell lines, peroxisome proliferator-activated receptor γ agonists (GW1929, rosiglitazone, pioglitazone) significantly decrease OCT-1 activity; conversely, peroxisome proliferator-activated receptor γ antagonists (GW9662, T0070907) increase OCT-1 activity. Importantly, these effects can lead to corresponding changes in sensitivity to BCR-ABL kinase inhibition. Results were confirmed in peroxisome proliferator-activated receptor γ-transduced K562 cells. Furthermore, we identified a strong negative correlation between OCT-1 activity and peroxisome proliferator-activated receptor γ transcriptional activity in diagnostic chronic myeloid leukemia patients (n=84; P<0.0001), suggesting that peroxisome proliferator-activated receptor γ activation has a negative impact on the intracellular uptake of imatinib and consequent BCR-ABL kinase inhibition. The inter-patient variability of peroxisome proliferator-activated receptor γ activation likely accounts for the heterogeneity observed in patient OCT-1 activity at diagnosis. Recently, the peroxisome proliferator-activated receptor γ agonist pioglitazone was reported to act synergistically with imatinib, targeting the residual chronic myeloid leukemia stem cell pool. Our findings suggest that peroxisome proliferator-activated receptor γ ligands have differential effects on circulating mononuclear cells compared to stem cells. Since the effect of peroxisome proliferator-activated receptor γ activation on imatinib uptake in mononuclear cells may counteract the clinical benefit of this activation in stem cells, caution should be applied when combining these therapies, especially in patients with high peroxisome proliferator-activated receptor γ transcriptional activity.

Oncogenic ZEB2 activation drives sensitivity toward KDM1A inhibition in T-cell acute lymphoblastic leukemia.

Elevated expression of the Zinc finger E-box binding homeobox transcription factor-2 (ZEB2) is correlated with poor prognosis and patient outcome in a variety of human cancer subtypes. Using a conditional gain-of-function mouse model, we recently demonstrated that ZEB2 is an oncogenic driver of immature T-cell acute lymphoblastic leukemia (T-ALL), a heterogenic subgroup of human leukemia characterized by a high incidence of remission failure or hematological relapse after conventional chemotherapy. Here, we identified the lysine-specific demethylase KDM1A as a novel interaction partner of ZEB2 and demonstrated that mouse and human T-ALLs with increased ZEB2 levels critically depend on KDM1A activity for survival. Therefore, targeting the ZEB2 protein complex through direct disruption of the ZEB2-KDM1A interaction or pharmacological inhibition of the KDM1A demethylase activity itself could serve as a novel therapeutic strategy for this aggressive subtype of human leukemia and possibly other ZEB2-driven malignancies.

MDR reversal activity of bromotetrandrine in vitro and in vivo.

The present study was aimed to evaluate the MDR reversal activity of bromotetrandrine (BrTet) in vitro and in vivo. The inhibitory effects of adriamycin (ADM) used alone or in combination with BrTet or Tet on the proliferation of K562 and K562/A02 cells were evaluated by MTT assay. The ADM accumulation and the protein levels of P-glycoprotein (P-gp) were detected by flow cytometry. The mRNA levels of P-gp were determined by RT-PCR. The in vivo effect of BrTet and Tet was investigated by using nude mice grafted with sensitive human leukemia cell line K562 and MDR cell line K562/A02. The results showed that BrTet at 0.25, 0.5 and 1 micromol/L reversed the resistance to ADM in MDR K562/A02 cells in a dose-dependent manner. Flow cytometry suggested that BrTet significantly increased the intracellular accumulation of ADM in K562/A02 cells in a dose-dependent manner. BrTet also inhibited the overexpression of P-gp in K562/A02 cells, and down-regulated mdr1 expression. In nude mice bearing K562 xenografts on the left flank and K562/A02 xenografts on the right flank, intraperitoneal injection of 10 mg/kg BrTet significantly enhanced the antitumor activity of ADM against K562/A02 xenografts with inhibitory rates of 26.1%, while ADM alone inhibited the growth of K562/A02 xenografts only by 5.8%. No enhancement effect by BrTet was seen in K562 xenografts. It is concluded that BrTet shows significant MDR reversal activity in vitro and in vivo. Its activity may be related to the inhibition of P-gp overexpression and the increase intracellular accumulation of anticancer drugs. BrTet may be a promising-MDR modulator for eventual assessment in the clinic.

[Comparison of reversal effects of 5-bromotetrandrine and tetrandrine on P-glycoprotein-dependent Resistance to adriamycin in human lukemia cell line K562/A02].

BACKGROUND & OBJECTIVE: 5-Bromotetrandrine (BrTet), a bromized derivative of tetrandrine (Tet), could effectively reverse P-glycoprotein (P-gp)-mediated multidrug resistance (MDR). This study was to compare the reversal effects of BrTet and Tet on MDR of human leukemia cell line K562/A02. METHODS: The effects of BrTet on the proliferation of K562 and K562/A02 cells were observed by MTT assay. The inhibitory effects of adriamycin (ADM) used alone or in combination with BrTet or Tet on the proliferation of K562 and K562/A02 cells were evaluated by MTT assay. The effect of BrTet or Tet on ADM accumulation was analyzed by flow cytometry (FCM). The protein level of P-gp was detected by Western blot. RESULTS: The inhibition rates of low concentrations of BrTet (< or =2.0 micromol/L) and Tet (< or =1.5 micromol/L) on the proliferation of K562 and K562/A02 cells were below 10%; no significant cytotoxicity was observed. The resistance of K562/A02 cells to ADM was 49.51 folds of that of K562 cells. When added 1.0 micromol/L Tet, the chemosensitivity of K562/A02 cells to ADM was increased to 12.17 folds; when added 0.25, 0.5 and 1.0 micromol/L BrTet, the chemosensitivity of K562/A02 cells to ADM was increased to 17.88, 9.9 and 4.24 folds, respectively. FCM showed that 1.0 micromol/L BrTet inhibited the overexpression of P-gp and increased the accumulation of ADM in K562/A02 cells, and its potency was greater than that of 1.0 micromol/L Tet(P<0.05). CONCLUSIONS: BrTet could reverse MDR in vitro. Its activity may be related to the inhibition of P-gp overexpression and the increase in intracellular accumulation of anticancer drugs.