Catalytic Mechanism Investigation of Lysine-Specific Demethylase 1 (LSD1): A Computational Study

Lysine-specific demethylase 1 (LSD1), the first identified histone demethylase, is a flavin-dependent amine oxidase which specifically demethylates mono- or dimethylated H3K4 and H3K9 via a redox process. It participates in a broad spectrum of biological processes and is of high importance in cell proliferation, adipogenesis, spermatogenesis, chromosome segregation and embryonic development. To date, as a potential drug target for discovering anti-tumor drugs, the medical significance of LSD1 has been greatly appreciated. However, the catalytic mechanism for the rate-limiting reductive half-reaction in demethylation remains controversial. By employing a combined computational approach including molecular modeling, molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations, the catalytic mechanism of dimethylated H3K4 demethylation by LSD1 was characterized in details. The three-dimensional (3D) model of the complex was composed of LSD1, CoREST, and histone substrate. A 30-ns MD simulation of the model highlights the pivotal role of the conserved Tyr761 and lysine-water-flavin motif in properly orienting flavin adenine dinucleotide (FAD) with respect to substrate. The synergy of the two factors effectively stabilizes the catalytic environment and facilitated the demethylation reaction. On the basis of the reasonable consistence between simulation results and available mutagenesis data, QM/MM strategy was further employed to probe the catalytic mechanism of the reductive half-reaction in demethylation. The characteristics of the demethylation pathway determined by the potential energy surface and charge distribution analysis indicates that this reaction belongs to the direct hydride transfer mechanism. Our study provides insights into the LSD1 mechanism of reductive half-reaction in demethylation and has important implications for the discovery of regulators against LSD1 enzymes

LncRNAs GIHCG and SPINT1-AS1 Are Crucial Factors for Pan-Cancer Cells Sensitivity to Lapatinib

Lapatinib is a small molecule inhibitor of EGFR (HER1) and ERBB2 (HER2) receptors, which is used for treatment of advanced or metastatic breast cancer. To find the drug resistance mechanisms of treatment for EGFR/ERBB2 positive tumors, we analyzed the possible effects of lncRNAs. In this study, using CCLE (Cancer Cell Line Encyclopedia) database, we explored the relationship between the lncRNAs and Lapatinib sensitivity/resistance, and then validated those findings through in vitro experiments. We found that the expression of EGFR/ERBB2 and activation of ERBB pathway was significantly related to Lapatinib sensitivity. GO (Gene Oncology) analysis of top 10 pathways showed that the sensitivity of Lapatinib was positively correlated with cell keratin, epithelial differentiation, and cell-cell junction, while negatively correlated with signatures of extracellular matrix. Forty-four differentially expressed lncRNAs were found between the Lapatinib sensitive and resistant groups (fold-change > 1.5, P < 0.01). Gene set variation analysis (GSVA) was performed based on 44 lncRNAs and genes in the top 10 pathways. Five lncRNAs were identified as hub molecules. Co-expression network was constructed by more than five lncRNAs and 199 genes in the top 10 pathways, and three lncRNAs (GIHCG, SPINT1-AS1, and MAGI2-AS3) and 47 genes were identified as close-related molecules. The three lncRNAs in epithelium-derived cancers were differentially expressed between sensitive and resistant groups, but no significance was found in non-epithelium-derived cancer cells. Correlation analysis showed that SPINT1-AS1 (R = −0.715, P < 0.001) and GIHCG (R = 0.557, P = 0.013) were correlated with the IC50 of epithelium-derived cancer cells. In further experiments, GIHCG knockdown enhanced cancer cell susceptibility to Lapatinib, while high level of SPINT1-AS1 was a sensitive biomarker of NCI-N87 and MCF7 cancer cells to Lapatinib. In conclusions, lncRNAs GIHCG and SPINT1-AS1 were involved in regulating Lapatinib sensitivity. Up-regulation of GIHCG was a drug-resistant biomarker, while up-regulation of SPINT1-AS1 was a sensitive indicator

Molecular Basis of NDM-1, a New Antibiotic Resistance Determinant

The New Delhi Metallo-β-lactamase (NDM-1) was first reported in 2009 in a Swedish patient. A recent study reported that Klebsiella pneumonia NDM-1 positive strain or Escherichia coli NDM-1 positive strain was highly resistant to all antibiotics tested except tigecycline and colistin. These can no longer be relied on to treat infections and therefore, NDM-1 now becomes potentially a major global health threat

FTY720 Induces Apoptosis of M2 Subtype Acute Myeloid Leukemia Cells by Targeting Sphingolipid Metabolism and Increasing Endogenous Ceramide Levels

<div><p>The M2 subtype Acute Myeloid Leukemia (AML-M2) with t(8;21) represents an unmet challenge because of poor clinical outcomes in a sizable portion of patients. In this study,we report that FTY720 (Fingolimod), a sphingosine analogue and an FDA approved drug for treating of multiple sclerosis, shows antitumorigenic activity against the Kasumi-1 cell line, xenograft mouse models and leukemic blasts isolated from AML-M2 patients with t(8;21) translocation. Primary investigation indicated that FTY720 caused cell apoptosis through caspases and protein phosphatase 2A (PP2A) activation. Transcriptomic profiling further revealed that FTY720 treatment could upregulate AML1 target genes and interfere with genes involved in ceramide synthesis. Treatment with FTY720 led to the elimination of AML1-ETO oncoprotein and caused cell cycle arrest. More importantly, FTY720 treatment resulted in rapid and significant increase of pro-apoptotic ceramide levels, determined by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry based lipidomic approaches. Structural simulation model had also indicated that the direct binding of ceramide to inhibitor 2 of PP2A (I2PP2A) could reactivate PP2A and cause cell death. This study demonstrates, for the first time, that accumulation of ceramide plays a central role in FTY720 induced cell death of AML-M2 with t(8;21). Targeting sphingolipid metabolism by using FTY720 may provide novel insight for the drug development of treatment for AML-M2 leukemia.</p></div

FTY720 induces elimination of AML1-ETO oncoprotein and causes cell cycle arrest.

<p>(A) Kasumi-1 cells were treated with indicated concentrations of FTY720 for 24 h, and cell lysate was subjected to western blotting analysis. Anti-ETO antibody was used to detect AML1-ETO fusion protein. (B) Kasumi-1 cells were treated with FTY720 for 72 hours, and cell cycle of Kasumi-1 cells treated FTY720 was analyzed by flow cytometry after propidium iodide (PI) staining, representative histograms were shown. (C) Cell cycle progression of Kasumi-1 treated with FTY720 was evaluated, nuclei stained with PI was used to DNA content analysis by flow cytometry. (D) Morphologic changes in Kasumi-1 treated for 3 days with indicated concentrations of FTY720. (E, F) Quantification of CD11b (E), CD11c (F), and CD117 expression in Kaumi-1 cells treated for 3 days with FTY720 was detected with flow cytometry.</p

<i>In vitro</i> efficacy of FTY720 on Kasumi-1 cells and fresh leukemic cells.

<p>(A) Kasumi-1 and SKNO-1 cells were treated with indicated concentration of FTY720 for 48 h, and the cell viability was determined with a CCK-8 kit. (B) Kasumi-1 cells were grown in methylcellulose for 14 d in presence of FTY720. **P<0.01, student's <i>t</i> test compared with untreated. (C) PBMCs from leukemia patients or healthy donor were treated with or without FTY720 for 24 h, and then subjected to apoptosis analysis by flow cytometry. *P<0.05, **P<0.01. (D) Kasumi-1 (top panel) and fresh leukemic cells (bottom panel) from AML-M2 patient were treated or untreated with FTY720 (7.5 µM), and analyzed by wright staining. Partial apoptotic cells were indicated by red arrows, and blue arrow presented cells might undergo differentiation.</p

FTY720 induces Kasumi-1 cell apoptosis primarily through elevating ceramide levels.

<p>(A, B) Ceramide levels in Kasumi-1 cells treated FTY720 with indicated concentrations (A) and different times (B) were determined by HPLC-ESI-MS/MS assay, TC means total ceramide (C14-, C16-, C18-, C20-, and C22-Ceramide). *P<0.05. (C) Kasumi-1 cells were treated with FTY720 for indicated times, and mitochondria was separated. The contents of ceramide in mitochondria of FTY720 treated Kasumi-1 cells were determined by HPLC-ESI-MS/MS assay. * P<0.05. (D) Kasumi-1 cells treated with or without Ara-C (7.5 µM) for 24 h, the contents of sphingolipid were determined by HPLC-ESI-MS/MS assay. (E, F) Kasumi-1 cells treated with indicated concentrations of FTY720 for 24 h, the contents of S1P (E) or SPH (F) were determined by HPLC-ESI-MS/MS assay. *P<0.05. (G) Kasumi-1 cells treated with indicated concentrations of FTY720 for 24 h, the signals of sphingolipids detected by HPLC-ESI-MS/MS assay were shown. (H) Kasumi-1 cells were pretreated with 20 µM FB1 or GW4869 for 3 h, and treated with 7.5 µM FTY720 for further 21 h. The effects of FB1 or GW4869 on FTY720-induced apoptosis were evaluated by flow cytometry. *P<0.05. (I) Kasumi-1 cells were pretreated with amitriptyline for 3 h, and treated with 7.5 µM FTY720 for further 21 h. The effects of amitriptyline on FTY720-mediated apoptosis were determined by flow cytometry.</p

FTY720 suppresses Kasumi-1 xenograft tumor growth.

<p>Nude mice (7 or 8 mice per group) with Kasumi-1 tumor were injected intraperitoneally with vehicle (5% glucose), Ara C (5 mg/kg, positive control) or FTY720 (1 mg/kg, 5 mg/kg) for 21days. (A) Tumor volumes were measured every three days. Data are expressed as mean volumes. *P<0.05. (B, C) Animals were sacrificed at day 21, and tumors were excised and weighed. Average (B) and representative (C) results are shown. *P<0.05. **P<0.01 (D) TUNEL assay was used to detect apoptotic cells in tumors of Kasumi-1 Xenograft models treated with vehicle, Ara-C (5 mg/kg), or FTY720 (5 mg/kg).</p

FTY720 results in caspase-dependent cell apoptosis.

<p>Western blotting assay was used to analyze the activation of caspases in Kasumi-1 cells treated with FTY720. Kasumi-1 cells were treated with indicated concentrations of FTY720 for 24 h. Caspase-3 (casp-3) and activated caspase-3 (c-casp-3) were shown by the same antibody blotting (A), caspase-8 (casp-8), active caspase-8 (c-casp-8), caspase-9 (casp-9) and active caspase-9 (c-casp-9) were analyzed by indicated antibodies blotting (B). (C) Kasumi-1 cells were pretreated with DEVD or VAD for 3 h, and subjected to 7.5 µM FTY720 for another 21 h. The effects of DEVD and VAD on FTY720-induced apoptosis were evaluated by flow cytometry. *P<0.05. (D) Kasumi-1 cells were treated with 7.5 µM FTY720 for indicated times, and then western blotting assay was carried out to analysis casp-3, casp-8, and casp-9 activation in Kasumi-1 cells treated with FTY720 for different time.</p

Gene expression profile analysis of FTY720 treated Kasumi-1 cells.

<p>(A) Volcano plots of gene expression differences for Kasumi-1 cells treated with FTY720 for 6 hours. Blue dots represent gene probes with <i>P</i> value <0.05 by t test and down-regulated fold change >1.5 (log₂FC<−0.58). Red dots represent gene probes with <i>P</i><0.05 and fold change >1.5 (log₂FC>0.58). Gray dots represent gene probes with <i>P</i>>0.05 or fold change <1.5 (B) Gene Set Enrichment Analysis of sphingolipid metabolism signature. The enrichment is depicted by FDR q value and NES. (C) Heat map of the core enrichment genes (subset of genes that contributes the most to the enrichment result) in sphingolipid metabolism. (D) Gene set enrichment analysis of AML1 transcription factor signature. The enrichment is depicted by FDR q value and NES. (E, F) Kasumi-1 cells were treated with 5 µM FTY720 for 6 h, and RNA was extracted from cells. qRT-PCR was used to analyzetranscription of genes of ceramide synthesis pathway (E) and AML1 targets (F). *P<0.05, **P<0.01.</p