Journal: PLoS ONE

Article Title: Dual inhibition of Fes and Flt3 tyrosine kinases potently inhibits Flt3-ITD + AML cell growth

doi: 10.1371/journal.pone.0181178

Figure Lengend Snippet: In vitro inhibitor specificity profiles with recombinant Fes and Flt3 kinases. In vitro kinase assays were performed with recombinant full-length Fes as well as wild-type (WT), ITD and D835Y forms of the Flt3 kinase domain (Z’-Lyte assay; see Materials and methods ). Each inhibitor was tested in triplicate over a range of concentrations with the ATP concentration set to the K m value for each kinase. IC 50 values were calculated by non-linear regression analysis of the resulting dose-response curves. Also included in the table are the IC 50 values for growth suppression by each compound in four AML cell lines, calculated from the dose-response curves shown in Fig 2 . Note that MV4-11, MOLM-13, and MOLM-14 are Flt3-ITD + , while THP-1 is wild-type for Flt3.

Article Snippet: The human AML cell lines MV4-11 (CRL-9591) and THP-1 (TIB-202) were obtained from the American Type Culture Collection (ATCC), while the AML cell lines MOLM-13 (ACC-544) and MOLM-14 (ACC-777) were obtained from the Leibniz-Institute DSMZ-German Collection.

Techniques: In Vitro, Recombinant, Concentration Assay, Kinase Assay

Journal: bioRxiv

Article Title: Ferroptosis response segregates small cell lung cancer (SCLC) neuroendocrine subtypes

doi: 10.1101/2020.07.11.198408

Figure Lengend Snippet: ( A ) schematic view of genes involved in extrinsic apoptosis, ( B ) necroptosis and ( C ) ferroptosis. ( D , E , G , H , J , K ) RNA-seq expression data in FPKM (fragments per kilobase of exon model per million reads mapped) from normal lung (n=22) and mostly chemo-naïve SCLC patient samples (n=67) were log2 transformed (+0.01) and plotted for relative expression of genes involved in extrinsic apoptosis ( D , E ) necroptosis ( G , H) and ferroptosis ( J , K) boxplot center line, mean; box limits, upper and lower quartile; whiskers min. to max. ( F ) The indicated human SCLC cell lines (n=7) and NSCLC line (H460) were treated with human izTRAIL for 24 h, cell viability was determined by Cell Titer Blue. ( I ) Cells were treated with TNF (T) [10 ng/ml]/zVAD (Z) [20 μM]/Smac mimetic (S) Birinapant [1 μM] for 24 h, cell death was quantified by propidium iodide (PI) uptake and flow cytometry. ( L ) Cells were treated with erastin at indicated concentrations for 24 h, cell viability was determined by Cell Titer Blue. Data are means +/− SEM of three independent experiments wherever not indicated otherwise.

Article Snippet: Human SCLC cell lines (H82, COR-L88, H524, H889, L303, H526, H2171, H1836, H1092) were grown in suspension in RPMI (GIBCO) and were obtained from ATCC ( https://www.lgcstandards-atcc.org/?geo_country=de ).

Techniques: RNA Sequencing, Expressing, Transformation Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Ferroptosis response segregates small cell lung cancer (SCLC) neuroendocrine subtypes

doi: 10.1101/2020.07.11.198408

Figure Lengend Snippet: ( A ) the indicated murine SCLC lines (n=3) were treated either with DMSO, RSL3 [1 μM], erastin [10 μM] alone or in combination with Ferrostatin-1 (Fer-1) [5 μM] for 24 h. Cells were washed, cultured for 5 days for recovery and stained with crystal violet. ( B, C, D, E ) Human H82 cells were treated as indicated: erastin [10 μM] +/− Ferrostatin-1 (Fer-1, 5 μM) +/− Deferoxamine (DFO) [100 μM], RSL3 [1 μM] +/− Fer-1 +/− DFO for 24 h. Cell death was determined by propidium iodide (PI) uptake and flow cytometry. ( F ) RP285.5 murine SCLC cells stably expressing Cas9 and the indicated control or gRNAs targeting GPX4 were cultured with or without Ferrostatin-1 (Fer-1) for 24 h. Cell death was quantified by propidium iodide (PI) uptake and flow cytometry. ( G ) protein extracts were obtained from cells as in ( F ) cultured in the presence of Fer-1 [5 μM]. ( H, I ) RP285.5 murine SCLC cells as in ( F ) were cultured in the presence or withdrawal of Fer-1 [5 μM] for 5 h and stained for lipid ROS accumulation using BODIPY C11. Cells were analyzed by flow cytometry and mean fluorescent intensity (MFI) was quantified. Data are means +/− SEM of three independent experiments in each individual cell line or representative images or histograms were applicable.

Article Snippet: Human SCLC cell lines (H82, COR-L88, H524, H889, L303, H526, H2171, H1836, H1092) were grown in suspension in RPMI (GIBCO) and were obtained from ATCC ( https://www.lgcstandards-atcc.org/?geo_country=de ).

Techniques: Cell Culture, Staining, Flow Cytometry, Stable Transfection, Expressing, Control

Journal: bioRxiv

Article Title: Ferroptosis response segregates small cell lung cancer (SCLC) neuroendocrine subtypes

doi: 10.1101/2020.07.11.198408

Figure Lengend Snippet: ( A , B ) Human SCLC cell lines (n=8) were treated as indicated for 24 h, cell viability was determined by Cell Titer Blue. ( C ) the indicated human SCLC cells were treated with DMSO, RSL3 [100 nM] or RSL3/Fer-1 [5 μM] for 5 h and stained for lipid ROS accumulation using BODIPY C11. Cells were analyzed by flow cytometry. ( D ) RNA-seq data of human SCLC lines (sensitive n=3, H524, NCIH82, H526; resistant n=5, NCIH889, NCIH1092, CORL88, H1836, H2171) were analyzed for differential expression between responders and non-responders, heatmap represents hierarchical clustering of FPKM (log2+0.01) of the 50 most differentially expressed genes. ( E ) ASCL1 and REST1 expression (FPKM (log2+0.01) comparing sensitive and resistant cells is plotted, boxplot center line, mean; box limits, upper and lower quartile; whiskers min. to max. ( F ) Gene set enrichment (GSEA) of a ranked list from ferroptosis sensitive and resistant cells was performed. ( G ) stickers and floaters were cultured separately; representative images were taken at 200x magnification. ( H ) western blot of SCLC subtype marker expression in manually separated stickers and floaters . ( I ) stickers and floaters were cultured separately and treated with RSL3 at the indicated concentrations for 24 h, cell viability was determined by Cell Titer Blue. ( J ) stickers and floaters were cultured separately and treated with DMSO, RSL3 [1 μM] +/− Fer-1 [5 μM] for 5 h and stained for lipid ROS accumulation using BODIPY C11 and analyzed by flow cytometry; mean fluorescent intensity (MFI) was quantified. ( K ) gating strategy for ASCL1 high stickers and floaters FACS sorting. ( L ) stickers and floaters were FACS-sorted by ASCL1 expression and ACSL4 protein levels were detected by Western blot. ( M ) Heatmap showing the representation of mono-oxidized phospholipid species (PE, phosphatidylethanolamine; PC, phosphatidylcholine) in stickers as compared to floaters treated with either DMSO or RSL3 [1 μM] for 5 h and then subjected to lipidomics. Samples for each condition (n=5) were averaged and normalized to the cell number (2.5×10 6 ). Each lipid species was normalized to levels detected in floaters +DMSO. One representative out of two independent experiments is shown. Data are means +/− SEM of three independent experiments or representative images if not indicated otherwise.

Article Snippet: Human SCLC cell lines (H82, COR-L88, H524, H889, L303, H526, H2171, H1836, H1092) were grown in suspension in RPMI (GIBCO) and were obtained from ATCC ( https://www.lgcstandards-atcc.org/?geo_country=de ).

Techniques: Staining, Flow Cytometry, RNA Sequencing, Quantitative Proteomics, Expressing, Cell Culture, Western Blot, Marker

Journal: bioRxiv

Article Title: Ferroptosis response segregates small cell lung cancer (SCLC) neuroendocrine subtypes

doi: 10.1101/2020.07.11.198408

Figure Lengend Snippet: ( A ) schematic view of genes involved in the TRX antioxidant pathway. ( B ) H82 and H2171 cells were lysed and TRX pathway component expression was detected by Western blot. ( C ) indicated cells were treated with Auranofin [1 μM] for the indicated times and subjected to redox shift assays. Densitometrical quantification of TRX redox forms is shown. ( D ) indicated cells were treated with Auranofin for 24 h, cell viability was determined by Cell Titer Blue. ( E ) indicated cells were treated with BSO for 24 h, cell viability was determined by Cell Titer Blue. ( F , G ) RP181.5 manually separated stickers and floaters were treated with Ferrostatin-1 [5 μM] for 2 h prior to adding DMSO, BSO [10 mM] or Auranofin [1 μM] for an additional 24h. DRAQ7 [0,1 μM] (red color in image) was added to all wells to visualize dead cells. Images were acquired every 5 h using the IncuCyte S3 bioimaging platform. Scale bar = 100 μm. Data are means +/− SEM of three independent experiments or representative images where applicable. ( H ) RP181.5 stickers were treated with either DMSO, Auranofin [500 nM] or BSO [500 μM] for 96 h and then fixed and stained for ASCL1 (red) and counterstained with DAPI (blue). Scale bar = 100 μm. ( I ) schematic view of SCLC NE subtype ferroptosis vulnerability and its effect in heterogeneous tumors. Data are means +/− SEM of three independent experiments or representative images where applicable.

Article Snippet: Human SCLC cell lines (H82, COR-L88, H524, H889, L303, H526, H2171, H1836, H1092) were grown in suspension in RPMI (GIBCO) and were obtained from ATCC ( https://www.lgcstandards-atcc.org/?geo_country=de ).

Techniques: Expressing, Western Blot, Staining

Journal: bioRxiv

Article Title: Ferroptosis response segregates small cell lung cancer (SCLC) neuroendocrine subtypes

doi: 10.1101/2020.07.11.198408

Figure Lengend Snippet: ( A , B ) 8-weeks old male nude mice were injected with 1.5×10 6 H82 ( A ) and H2171 ( B ) cells into flanks. Once palpable, tumors were treated either with vehicle (H82, n=9; H2171 n=4) or combined BSO [5 mM] in the drinking water and Auranofin 3x per week i.p. [2.5 mg/kg] (H82, n=11, H2171 n=4) for two consecutive weeks. Fold change of initial tumor size is shown. ( C ) Sections from paraffin-embedded tumors of vehicle or combo-treated mice were stained by H&E or for MDA. Representative images are shown, scale bar=200 μm. Data are means +/− SEM of three independent experiments or representative images where applicable. ( D ) lung tumor volumes were quantified by Horos software using MRI scans. Mice were randomized to obtain two groups with equal mean tumor volume (n=8 for vehicle, n=7 for combo). (E) tumor-bearing RP-mice were treated either with vehicle (n=8) or combined BSO [5 mM] in the drinking water and Auranofin 3x per week i.p. [2.5 mg/kg] (n=7) for two consecutive weeks. Fold change in tumor volumes was determined by quantifying initial tumor volume from MRI scans as compared to tumor volume at the end of the treatment cycle using Horos software. ( F ) representative MRI images pre and post treatment of mice as in ( E ). ( G ) isolation scheme of human CDXs. ( H ) Cellular morphology of human CDXs. ( I ) CDXs (n=2) or healthy donor PBMCs (n=2) were treated with DMSO, BSO [PBMCs, 500 μM; CDXs, 50 μM], Auranofin [250 nM] or BSO [PBMCs, 500 μM; CDXs, 50 μM] /Auranofin [250 nM] for 24 h, cell viability was quantified by Cell Titer Blue (CDXs) or flow cytometric quantification of propidium iodide (PI)-negative cells (PBMCs). ( J ) Kaplan-Meier survival curves for SCLC patients (n=77) containing low (low 1/3 n=25, median survival 33 months) or high (high 2/3 n=52, median survival 22.5 months) expression of GPX4 mRNA. ( K ) as in ( J ) expression of TXNRD1 mRNA was correlated using the same cut-off (low=1/3, median survival 38 months; high 2/3, median survival 22.5 months). ( L ) Kaplan-Meier survival curves for SCLC patients with combined low or high GPX4 and TXNRD1 mRNA expression (low/low n=10, median survival 43.5 months; high/high n=16, median survival 16 months). Data are means+/− SEM were applicable ( E ).

Article Snippet: Human SCLC cell lines (H82, COR-L88, H524, H889, L303, H526, H2171, H1836, H1092) were grown in suspension in RPMI (GIBCO) and were obtained from ATCC ( https://www.lgcstandards-atcc.org/?geo_country=de ).

Techniques: Injection, Staining, Software, Isolation, Expressing

Journal: Biomedicines

Article Title: Asparagine synthetase (ASNS) Drives Tumorigenicity in Small Cell Lung Cancer

doi: 10.3390/biomedicines13123087

Figure Lengend Snippet: ASNS is required for SCLC development. ( A , B ) Immunoblots for ASNS in mouse SCLC cells and human SCLC and NSCLC cell lines. β-actin and GAPDH are used as a loading control. ( C ) Representative H&E-stained sections of tumors (left) derived from Rb / p53 / p130 ( RPP ) mice and immunostaining for ASNS (middle; red) and DAPI (right; blue). ( D ) Whole-mount X-gal-stained lungs (left) from Rb / p53 / p130 / Asns lox /+ ( RPP ) mice 9 months after Ad-Cre infection. The interior of cut lungs is shown, and arrows and arrowheads indicate tumors and small lesions, respectively. H&E-stained section of tumors (middle) and immunostaining for CGRP marking neuroendocrine cells (right; green). Scale bars, ( C ) 50 µm; ( D ) 5 mm (left), 200 µm (middle and right).

Article Snippet: A panel of Human SCLC cell lines (NCI-H524, NCI-H82, NCI-H69, NCI-H209, NCI-H1184, NCI-H2141, NCI-H2171, NCI-A549, NCI-H1650, and NCI-H2009) were kindly provided by multiple investigators; Adi Gazdar and John Minna (UT Southwestern Medical Center, Dallas, TX, USA), Julien Sage (Stanford University, Stanford, CA, USA), Hisashi Harada (Virginia Commonwealth University, Richmond, VA, USA), and Christopher Vakoc (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA).

Techniques: Western Blot, Control, Staining, Derivative Assay, Immunostaining, Infection

Journal: Biomedicines

Article Title: Asparagine synthetase (ASNS) Drives Tumorigenicity in Small Cell Lung Cancer

doi: 10.3390/biomedicines13123087

Figure Lengend Snippet: ASNS induces tumorigenic progression of SCLC cells. ( A – E ) Results of MTT assay measuring the viability of cells treated with ASNS knockdown for 8 days. mSCLC-1 and mSCLC-2 are mouse SCLC cells, and H524, H82, and H69 are human SCLC cell lines. MTT assays were repeated with similar results at least once. **, p < 0.001. Statistical tests were performed using an unpaired t test. Error bar, SD.

Article Snippet: A panel of Human SCLC cell lines (NCI-H524, NCI-H82, NCI-H69, NCI-H209, NCI-H1184, NCI-H2141, NCI-H2171, NCI-A549, NCI-H1650, and NCI-H2009) were kindly provided by multiple investigators; Adi Gazdar and John Minna (UT Southwestern Medical Center, Dallas, TX, USA), Julien Sage (Stanford University, Stanford, CA, USA), Hisashi Harada (Virginia Commonwealth University, Richmond, VA, USA), and Christopher Vakoc (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA).

Techniques: MTT Assay, Knockdown

Journal: Biomedicines

Article Title: Asparagine synthetase (ASNS) Drives Tumorigenicity in Small Cell Lung Cancer

doi: 10.3390/biomedicines13123087

Figure Lengend Snippet: ASNS is required for the expansion of human SCLC lines and mouse tumor cells. ( A – H ) Representative images (left) and quantification (right) of soft agar colonies formed by mouse and human SCLC cell lines (n = 3 per cell type). *, p < 0.01; **, p < 0.001. Statistical tests were performed using an unpaired t test. Error bar, SD. Scale bars, ( A , C , E , G ), 5 mm.

Article Snippet: A panel of Human SCLC cell lines (NCI-H524, NCI-H82, NCI-H69, NCI-H209, NCI-H1184, NCI-H2141, NCI-H2171, NCI-A549, NCI-H1650, and NCI-H2009) were kindly provided by multiple investigators; Adi Gazdar and John Minna (UT Southwestern Medical Center, Dallas, TX, USA), Julien Sage (Stanford University, Stanford, CA, USA), Hisashi Harada (Virginia Commonwealth University, Richmond, VA, USA), and Christopher Vakoc (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA).

Techniques: