Extracellular vesicles of Bacteroides fragilis regulated macrophage polarization through promoted Sema7a expression.

Abnormal activation of macrophage and gut Bacteroides fragilis (BF) are the important induction factors in the occurrence of type 2 diabetes (T2D) and vascular complications. However, it remains unknown whether BF involves in macrophage polarization. In this study, we found that BF extracellular vesicles (EV) can be uptaken by macrophage. BF-EV promote macrophage M1/M2 polarization significantly, and increase Sting expression significantly. Bioinformatics analysis found that Sema7a is an important gene involving in macrophage polarization. The expression of Sema7a can be induced by BF-EV and can be inhibited after C-176 treated. The inhibition expression of Sema7a prevent BF-EV to induce macrophage polarization. Further analysis reveals that there is no direct interaction between Sting and Sema7a, but Sgpl1 can interact with Sting or Sema7a. BF-EV promote the expression of Sgpl1, which the phenomenon can be inhibited after C-176 treated. Importantly, overexpression of Sgpl1 reversed the effect of C-176 for Sema7a expression, while inhibit Sema7a expression has limitation influence for Sting and Sgpl1 expression. In conclusion, this study confirms that Sting-Sgpl1-Sema7a is a key mechanism by which BF-EV regulates macrophage polarization.

Pravastatin promotes type 2 diabetes vascular calcification through activating intestinal Bacteroides fragilis to induce macrophage M1 polarization.

BACKGROUND: Pravastatin is an oral lipid-lowering drug, commonly used by patients with diabetes that is positively correlated with the occurrence of vascular calcification (VC), but the mechanism is unclear. METHODS: In this study, 16S rRNA sequencing and qRT-PCR wereused to detect the differential gut bacteria. Metabolomics and ELISA were used to analyze the differential metabolites. qRT-PCR and western blotting (WB) were used to detect genes expression. Flow cytometry was used to analyze macrophage phenotype. Immunohistochemistry was used to analyze aortic calcification. RESULTS: We found that gut Bacteroides fragilis (BF) increased significantly in patients who took pravastatin or type 2 diabetes (T2D) mice treated with pravastatin. In vitro experiments showed that pravastatin had little effect on BF but significantly promoted BF proliferation in vivo. Further analysis showed that ArsR was an important gene for pravastatin to regulate the activation of BF, and overexpression of ArsR significantly promoted the secretion of 3,4,5-trimethoxycinnamic acid (TMCA). Importantly, pravastatin significantly promoted BF secretion of TMCA and significantly increased TMCA secretion in T2D patients or T2D mice. TMCA had little effect on vascular smooth muscle cell calcification but significantly promoted macrophage M1 polarization, which we had demonstrated that M1 macrophages promoted T2D VC. In vivo studies found that pravastatin significantly upregulated TMCA levels in the feces and serum of T2D mice transplanted with BF and promoted the macrophage M1 polarization in bone marrow and the osteoblastic differentiation of aortic cells. Similar results were obtained in T2D mice after intravenous infusion of TMCA. CONCLUSIONS: Promoting intestinal BF to secrete TMCA, which leads to macrophage M1 polarization, is an important mechanism by which pravastatin promotes calcification, and the result will be used for the optimization of clinical medication strategies of pravastatin supplying a theoretical basis and experimental basis.

Emerging role of RNA acetylation modification ac4C in diseases: Current advances and future challenges.

The oldest known highly conserved modification of RNA, N4-acetylcytidine, is widely distributed from archaea to eukaryotes and acts as a posttranscriptional chemical modification of RNA, contributing to the correct reading of specific nucleotide sequences during translation, stabilising mRNA and improving transcription efficiency. Yeast Kre33 and human NAT10, the only known authors of ac4C, modify tRNA with the help of the Tan1/THUMPD1 adapter to stabilise its structure. Currently, the mRNA for N4-acetylcytidine (ac4C), catalysed by NAT10 (N-acetyltransferase 10), has been implicated in a variety of human diseases, particularly cancer. This article reviews advances in the study of ac4C modification of RNA and the ac4C-related gene NAT10 in normal physiological cell development, cancer, premature disease and viral infection and discusses its therapeutic promise and future research challenges.

Amnio acid substitution at position 298 of human glucose-6 phosphatase-α significantly impacts its stability in mammalian cells.

Glucose-6-phosphatase-α (G6Pase-α) catalyzes the hydrolysis of glucose-6-phosphate to glucose and functions as a key regulator in maintaining blood glucose homeostasis. Deficiency in G6Pase-α causes glycogen storage disease 1a (GSD1a), an inherited disorder characterized by life-threatening hypoglycemia and other long-term complications. We have developed a potential mRNA-based therapy for GSD1a and demonstrated that a human G6Pase-α (hG6Pase-α) variant harboring a single serine (S) to cysteine (C) substitution at the amino acid site 298 (S298C) had > twofold increase in protein expression, resulting in improved in vivo efficacy. Here, we sought to investigate the mechanisms contributing to the increased expression of the S298C variant. Mutagenesis of hG6Pase-α identified distinct protein variants at the 298 amino acid position with substantial reduction in protein expression in cultured cells. Kinetic analysis of expression and subcellular localization in mammalian cells, combined with cell-free in vitro translation assays, revealed that altered protein expression stemmed from differences in cellular protein stability rather than biosynthetic rates. Site-specific mutagenesis studies targeting other cysteines of the hG6Pase-α S298C variant suggest the observed improvements in stability are not due to additional disulfide bond formation. The glycosylation at Asparagine (N)-96 is critical in maintaining enzymatic activity and mutations at position 298 mainly affected glycosylated forms of hG6Pase-α. Finally, proteasome inhibition by lactacystin improved expression levels of unstable hG6Pase-α variants. Taken together, these data uncover a critical role for a single amino acid substitution impacting the stability of G6Pase-α and provide insights into the molecular genetics of GSD1a and protein engineering for therapeutic development.

Extracellular vesicle miR-32 derived from macrophage promotes arterial calcification in mice with type 2 diabetes via inhibiting VSMC autophagy.

BACKGROUND: The development of diabetes vascular calcification (VC) is tightly associated with the inhibition of vascular smooth muscle cell (VSMC) autophagy. Previously, our team found that miR-32-5p (miR-32) promotes macrophage activation, and miR-32 is expressed at higher level in the plasma of patients with coronary calcification. However, whether miR-32 mediates the function of macrophages in type 2 diabetes (T2D) VC is still unclear. METHODS: Wild-type (WT) and miR-32-/- mice were used in this study. qRT-PCR and western blotting were used to analyze gene expression. Flow cytometry was used to analyze the influence of glucose concentration on macrophage polarization. Nanoparticle tracking analysis (NTA), transmission electron microscopy, and confocal microscopy were used to identify macrophage extracellular vehicles (EVs). Immunofluorescence, in situ hybridization (ISH), immunohistochemistry, and alizarin red staining were used to analyze the influence of macrophage EVs on autophagy and calcification of the aorta of miR-32-/- mice. A luciferase assay was used to analyze the effect of miR-32 on myocyte enhancer factor 2D (Mef2d) expression. Co-IP combined with mass spectrometry (MS) and transcriptome sequencing was used to analyze the signalling pathway by which Mef2d acts in VSMC autophagy. RESULTS: We found that high glucose conditions upregulate miR-32 expression in macrophages and their EVs. Importantly, macrophages and their EVs promote VSMC osteogenic differentiation and upregulate miR-32 expression in VSMCs. Moreover, miR-32 mimics transfection promoted osteogenic differentiation and inhibited autophagy in VSMCs. In vitro and in vivo experiments showed that Mef2d is the key target gene of miR-32 that inhibits VSMC autophagy. Furthermore, MS and transcriptome sequencing found that cGMP-PKG is an important signalling pathway by which Mef2d regulates VSMC autophagy. In addition, after T2D miR-32-/- mice were injected with macrophage EVs via the caudal vein, miR-32 was detected in aortic VSMCs of miR-32-/- mice. Moreover, autophagy was significantly inhibited, and calcification was significantly enhanced in aorta cells. CONCLUSIONS: These results reveal that EVs are the key pathway by which macrophages promote T2D VC, and that EVs miR-32 is a key cause of autophagy inhibition in VSMCs.

The roles of immune cells in atherosclerotic calcification.

Atherosclerosis is the leading cause of acute cardiovascular events, and vascular calcification is an important pathological phenomenon in atherosclerosis. Recently, many studies have shown that immune cells are closely associated with the development of atherosclerosis and calcification, but there are many conflicting viewpoints because of immune system complications, such as the pro-atherosclerotic and atheroprotective effects of regulatory B cells (Bregs), T helper type 2 (Th2) cells and T helper type 17 (Th17) cells. In this review, we summarize the studies on the roles of immune cells, especially lymphocytes and macrophages, in atherosclerotic calcification. Furthermore, we prepared graphs showing the relationship between T cells, B cells and macrophages and atherosclerotic calcification. Finally, we highlight some potential issues that are closely associated with the function of immune cells in atherosclerotic calcification. Based on current research results, this review summarizes the relationship between immune cells and atherosclerotic calcification, and it will be beneficial to understand the relationship of immune cells and atherosclerotic calcification.

Tumor-associated macrophages: An important player in breast cancer progression.

Breast cancer is the most common form of malignant tumor in females, accounting for the second highest mortality among cancer patients. In the breast tumor microenvironment, tumor-associated macrophages (TAMs) are the most abundant immune cells, which regulate the progression of breast cancer. During breast cancer tumorigenesis and progression, TAMs support breast tumor growth by promoting angiogenesis and cancer cell metastasis, inducing cancer stemness, regulating energy metabolism, and supporting immune system suppression. TAMs exhibit a high degree of cellular plasticity. Repolarizing tumor-related macrophages into M1 macrophages can promote tumor regression. This study reviews the role and mechanism of action of TAMs in the development of breast cancer and establishes TAMs as effective targets for breast cancer treatment.

Preparation and application of modified three-dimensional cellulose microspheres for paclitaxel targeted separation.

In this article, we successfully prepared three-dimensional cellulose microspheres modified by molecularly imprinted polymer for paclitaxel recognition and separation (3D-CM &PTX&MIPs). The material was characterized by Fourier Transform Infrared spectroscopy (FTIR), Scanning Electron Microscope (SEM), Thermogravimetric Analysis (TG) and diffraction of X-rays (XRD). Under the optimized adsorption conditions, the maximum adsorption capacity reached 65.7 mg/g. And after 5 runs of reuse, (3D-CM&PTX&MIPs) still maintained a reusability rate of 90%. Besides, (3D-CM&PTX&MIPs) showed excellent selectivity for target PTX. Finally, (3D-CM&PTX&MIPs) was used for PTX recognition and separation in the extracts of yew leaves. This research laid a good foundation and scientific basis for the efficient, environmentally friendly, and rapid enrichment of metabolites in plants using bio-based molecularly imprinted polymers.

A cellulose-based temperature sensitivity molecular imprinted hydrogel for specific recognition and enrichment of paclitaxel.

A microcrystalline cellulose-based temperature sensitivity paclitaxel molecular imprinted hydrogel (MCC-TSMIHs-PTX) was successfully prepared by temperature-sensitive monomer N-isopropylacrylamide, functional monomer 4-vinylpyridine, cross-linking agent N, N'-methylenebisacrylamide and microcrystalline cellulose. They showed imprinting effective responses to the temperature changes. The results of adsorption kinetics, adsorption equilibrium, thermodynamics, selectivity and reusability showed the successful formation of a grafting thermosensitivity hydrogel with higher adsorption capacity and specific recognition. When the temperature reached 308 K, imprinting effect of hydrogel cavities would be most effective and conducive to capture template molecules. When the temperature reached 288 K, the lowest imprinting effect would facilitate the desorption of PTX. Finally, the MCC-TSMIHs-PTX was applied to enrich the paclitaxel in Taxus × media extracts samples, the relative contents of PTX in the samples were increased greatly from 7.23% to 78.32%, indicating the MCC-TSMIHs-PTX was a stable adsorption capacity for efficient separation and enrichment of PTX in Taxus × media extracts.

mRNA therapy restores euglycemia and prevents liver tumors in murine model of glycogen storage disease.

Glycogen Storage Disease 1a (GSD1a) is a rare, inherited metabolic disorder caused by deficiency of glucose 6-phosphatase (G6Pase-α). G6Pase-α is critical for maintaining interprandial euglycemia. GSD1a patients exhibit life-threatening hypoglycemia and long-term liver complications including hepatocellular adenomas (HCAs) and carcinomas (HCCs). There is no treatment for GSD1a and the current standard-of-care for managing hypoglycemia (Glycosade®/modified cornstarch) fails to prevent HCA/HCC risk. Therapeutic modalities such as enzyme replacement therapy and gene therapy are not ideal options for patients due to challenges in drug-delivery, efficacy, and safety. To develop a new treatment for GSD1a capable of addressing both the life-threatening hypoglycemia and HCA/HCC risk, we encapsulated engineered mRNAs encoding human G6Pase-α in lipid nanoparticles. We demonstrate the efficacy and safety of our approach in a preclinical murine model that phenotypically resembles the human condition, thus presenting a potential therapy that could have a significant therapeutic impact on the treatment of GSD1a.

Synthetic human ABCB4 mRNA therapy rescues severe liver disease phenotype in a BALB/c.Abcb4-/- mouse model of PFIC3.

BACKGROUND & AIMS: Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare lethal autosomal recessive liver disorder caused by loss-of-function variations of the ABCB4 gene, encoding a phosphatidylcholine transporter (ABCB4/MDR3). Currently, no effective treatment exists for PFIC3 outside of liver transplantation. METHODS: We have produced and screened chemically and genetically modified mRNA variants encoding human ABCB4 (hABCB4 mRNA) encapsulated in lipid nanoparticles (LNPs). We examined their pharmacological effects in a cell-based model and in a new in vivo mouse model resembling human PFIC3 as a result of homozygous disruption of the Abcb4 gene in fibrosis-susceptible BALB/c.Abcb4-/- mice. RESULTS: We show that treatment with liver-targeted hABCB4 mRNA resulted in de novo expression of functional hABCB4 protein and restored phospholipid transport in cultured cells and in PFIC3 mouse livers. Importantly, repeated injections of the hABCB4 mRNA effectively rescued the severe disease phenotype in young Abcb4-/- mice, with rapid and dramatic normalisation of all clinically relevant parameters such as inflammation, ductular reaction, and liver fibrosis. Synthetic mRNA therapy also promoted favourable hepatocyte-driven liver regeneration to restore normal homeostasis, including liver weight, body weight, liver enzymes, and portal vein blood pressure. CONCLUSIONS: Our data provide strong preclinical proof-of-concept for hABCB4 mRNA therapy as a potential treatment option for patients with PFIC3. LAY SUMMARY: This report describes the development of an innovative mRNA therapy as a potential treatment for PFIC3, a devastating rare paediatric liver disease with no treatment options except liver transplantation. We show that administration of our mRNA construct completely rescues severe liver disease in a genetic model of PFIC3 in mice.

A porous cellulose-based molecular imprinted polymer for specific recognition and enrichment of resveratrol.

A novel resveratrol molecularly imprinted polymer (p-CM@MPS@MIP-Res) was prepared on the surface of silanized porous cellulose microspheres (p-CM@MPS) for the first time, and was successfully applied for the efficient enrichment of targeted resveratrol in Polygonum cuspidatum. The adsorption kinetics and adsorption equilibrium of p-CM@MPS@MIP-Res were also studied in detail. Compared with non-molecularly imprinted polymer (p-CM@MPS@NIP), the prepared p-CM@MPS@MIP-Res showed high adsorption capacity for resveratrol, the adsorption capacity of the p-CM@MPS@MIP-Res could reach to 11.56 mg/g. Furthermore, the stability of the p-CM@MPS@MIP-Res was evaluated and the result showed that the p-CM@MPS@MIP-Res could be reused for 5 runs. Finally, the p-CM@MPS@MIP-Res was applied to enrich the resveratrol in Polygonum cuspidatum sample, the content of resveratrol in the extraction solution could be increased greatly from 4.23 % to 23.74 %, indicating the p-CM@MPS@MIP-Res was a promising adsorbent for efficiently separation and enrichment of resveratrol in Polygonum cuspidatum.

miR32-5p promoted vascular smooth muscle cell calcification by upregulating TNFα in the microenvironment.

BACKGROUND: Vascular calcification is often associated with chronic inflammation and is a risk factor for brain arterial stiffness. Our previous results showed that miR32-5p was positively correlated with vascular smooth muscle cells (VSMC) calcification, but it is unclear whether miR32-5p promoted VSMC calcification by regulating inflammatory factor production. RESULTS: In this study, bioinformatics analysis was used to select tumour necrosis factor α (TNFα) as a candidate inflammatory factor associated with calcification. Moreover, alizarin red staining and qRT-PCR analysis revealed that TNFα produced by BV2 cells was the key promoting factor of VSMC calcification. Interestingly, the expression of TNFα was significantly increased at the mRNA and protein levels after miR32-5p mimic treatment but significantly decreased after miR32-5p antagomir treatment. To explore the mechanism of the regulation of TNFα expression by miR32-5p, bioinformatics analysis indicated that PIKfyve was a candidate target gene of miR32-5p, and luciferase assays verified that the expression of PIKfyve was significantly repressed by miR32-5p mimics. Importantly, rescue experiments showed that the expression of TNFα in BV2 cells treated with miR32-5p antagomir and the PIKfyve inhibitor YM201636 was significantly increased. CONCLUSIONS: The production of TNFα in microglia could be affected by miR32-5p targeting PIKfyve, and these results will be beneficial to reveal the mechanism of brain arterial calcification.

Identification of an activation-related protein in B cells in the ABO incompatible condition.

In ABO-incompatible (ABOi) kidney transplantation (KT), antibodies can mediate immunological accommodation or immune rejection, but the mechanism by which B cells are induced to produce antibodies with different functions is still unclear. Previous research established an ABOi kidney cell model and identified that haptoglobin (HP) is associated with the activation of lymphocytes. In the present study, the results of a flow cytometric assay demonstrated that HP was expressed by B cells. Moreover, dot-ELISA and ELISA analyses showed that the concentrations of total IgG, blood group B antibody, IgG1, IgG2 and IgG4 were all significantly increased in the cell model. In addition, dot-ELISA and haptoglobin level analyses showed that HP protein expression was significantly increased, while RT-qPCR assay indicated that HP was significantly reduced at the mRNA level. Furthermore, bioinformatics analysis showed that HP could interact with Smad3, and the HP-Smad3 complex was detected in a peripheral blood mononuclear cell (PBMC) protein extract by a dot-ELISA method. This research revealed that HP was involved in the process of B-cell activation by interacting with Smad3, and the results will be helpful to reveal the mechanism of B-cell activation in ABOi-KT.

mRNA Therapy Improves Metabolic and Behavioral Abnormalities in a Murine Model of Citrin Deficiency.

Citrin deficiency is an autosomal recessive disorder caused by loss-of-function mutations in SLC25A13, encoding the liver-specific mitochondrial aspartate/glutamate transporter. It has a broad spectrum of clinical phenotypes, including life-threatening neurological complications. Conventional protein replacement therapy is not an option for these patients because of drug delivery hurdles, and current gene therapy approaches (e.g., AAV) have been hampered by immunogenicity and genotoxicity. Although dietary approaches have shown some benefits in managing citrin deficiency, the only curative treatment option for these patients is liver transplantation, which is high-risk and associated with long-term complications because of chronic immunosuppression. To develop a new class of therapy for citrin deficiency, codon-optimized mRNA encoding human citrin (hCitrin) was encapsulated in lipid nanoparticles (LNPs). We demonstrate the efficacy of hCitrin-mRNA-LNP therapy in cultured human cells and in a murine model of citrin deficiency that resembles the human condition. Of note, intravenous (i.v.) administration of the hCitrin-mRNA resulted in a significant reduction in (1) hepatic citrulline and blood ammonia levels following oral sucrose challenge and (2) sucrose aversion, hallmarks of hCitrin deficiency. In conclusion, mRNA-LNP therapy could have a significant therapeutic effect on the treatment of citrin deficiency and other mitochondrial enzymopathies with limited treatment options.

Salinomycin triggers endoplasmic reticulum stress through ATP2A3 upregulation in PC-3 cells.

BACKGROUND: Salinomycin is a monocarboxylic polyether antibiotic and is a potential chemotherapy drug. Our previous studies showed that salinomycin inhibited cell growth and targeted CSCs in prostate cancer. However, the precise target of salinomycin action is unclear. METHODS: In this work, we analyzed and identified differentially expressed genes (DEGs) after treatment with or without salinomycin using a gene expression microarray in vitro (PC-3 cells) and in vivo (NOD/SCID mice xenograft model generated from implanted PC-3 cells). Western blotting and immunohistochemical staining were used to analyze the expression of ATP2A3 and endoplasmic reticulum (ER) stress biomarkers. Flow cytometry was used to analyze the cell cycle, apoptosis and intracellular Ca2+ concentration. RESULTS: A significantly upregulated gene, ATPase sarcoplasmatic/endoplasmatic reticulum Ca2+ transporting 3 (ATP2A3), was successfully identified. In subsequent studies, we found that ATP2A3 overexpression could trigger ER stress and exert anti-cancer effects in PC-3 and DU145 cells. ATP2A3 was slightly expressed, but the ER stress biomarkers showed strong staining in prostate cancer tissues. We also found that salinomycin could trigger ER stress, which might be related to ATP2A3-mediated Ca2+ release in PC-3 cells. Furthermore, we found that salinomycin-triggered ER stress could promote apoptosis and thus exert anti-cancer effects in prostate cancer cells. CONCLUSION: This study demonstrates that ATP2A3 might be one of the potential targets for salinomycin, which can inhibit Ca2+ release and trigger ER stress to exert anti-cancer effects.

Salinomycin-induced autophagy blocks apoptosis via the ATG3/AKT/mTOR signaling axis in PC-3 cells.

AIMS: This study evaluated the mechanism by which salinomycin-induced autophagy blocks apoptosis in PC-3 prostate cancer cells. MAIN METHODS: The anti-cancer effects of salinomycin in PC-3 cells were confirmed by flow cytometry, JC-1 staining and western blotting. Then, the autophagic effects were measured by western blotting, GFP-LC3 puncta formation assay, immunofluorescence staining and electron microscopy. Furthermore, we used lentivirus-mediated shRNA to silence ATG3, ATG5 and ATG7 expression in PC-3 cells to investigate the regulatory mechanisms of salinomycin-induced autophagy. KEY FINDINGS: Salinomycin could induce apoptosis and autophagy in PC-3 cells. Interestingly, autophagy inhibition could enhance salinomycin-induced apoptosis. We further showed that ATG3, a known critical regulator of autophagy, was downregulated and involved in the inhibition of apoptosis by salinomycin-induced autophagy via the AKT/mTOR signaling axis. SIGNIFICANCE: Our data indicated that salinomycin-induced autophagy blocks apoptosis via the ATG3/AKT/mTOR signaling axis in PC-3 cells, which provides new clues for the mechanisms of underlying the anti-cancer effects of salinomycin.

The effects of hemocyanin on T cells cultured in vitro.

As a broad-spectrum antibiotic, gentamicin is used extensively in T cell culturing in vitro, but preliminary studies have identified that T cell activity is significantly affected by gentamicin. In the present study, the hemocyanin from Litopenaeus vannamei (L. vannamei) was selected as an additive for T cell cultures in vitro. Compared with those in the control group, the cell quantity exhibited no significant difference, and the formation rate of cell colony increased gradually with increases in the hemocyanin concentration. Additionally, flow cytometry assays identified that cluster of differentiation (CD)3+CD4+ and CD4+CD25+ T cells in the 0.2 µg/ml hemocyanin (Hem 3) group were all significantly increased. Furthermore, cell cycle analysis demonstrated that T cells in the G0/G1 phase were significantly decreased in the Hem 3 group compared with in the control, 0.05 µg/ml (Hem 1) and 0.1 µg/ml (Hem 2) groups, and cells in the S phase were significantly elevated in the Hem 3 group compared with in the control and Hem 1 groups. In addition, MTT analysis indicated that the cytotoxicity of T cells towards HepG2 cells was significantly increased in the Hem 3 group compared with in the control, Hem 1 and Hem 2 groups. Taken together, the present study identified that hemocyanin may improve the proliferation and cytotoxicity of T cells, and the results supported the use of hemocyanin in T cell adoptive immunotherapy.

Discovery of EBI-2511: A Highly Potent and Orally Active EZH2 Inhibitor for the Treatment of Non-Hodgkin's Lymphoma.

A novel series of benzofuran derived EZH2 inhibitors were discovered through a scaffold hopping approach based on the clinical compound of EPZ-6438. Further rational structure-activity relationship exploration and optimization led to the discovery of more potent EZH2 inhibitors with oral bioavailability in mice and rats. A lead compound EBI-2511 (compound 34) demonstrated excellent in vivo efficacy in Pfeiffer tumor Xenograft models in mouse and is under preclinical development for the treatment of cancers associated with EZH2 mutations.