hcc-15 acc-496  (DSMZ)

Journal: Nature Communications

Article Title: Targeting chromosomally unstable tumors with a selective KIF18A inhibitor

doi: 10.1038/s41467-024-55300-z

Figure Lengend Snippet: a Structure of VLS-1272. b Potency of VLS-1272 in a KIF18A (1-374) biochemical ADP-Glo assay to measure ATPase activity with varied concentrations of ATP. Data presented as mean values from n = 2 biological replicates. c Inhibition of KIF18A (1-374) by VLS-1272 as measured by ADP-Glo assay in the absence or presence of 0.1 mg/ml microtubules. Data presented as individual values from n = 2 biological replicates. d Representative still images from microtubule gliding filament assays with purified KIF18A 1-480-GFP linked to the glass surface in absence (control) or presence of VLS-1272. Arrows indicate the ends of microtubules that move directionally (control) or remain stationary (VLS-1272). e Plot of microtubule velocities measured in gliding filament assays in the presence or absence of VLS-1272. n = 249 (control) and 177 (VLS-1272) microtubules from 3 independent experiments. Data were compared using an unpaired, two-tailed t -test. **** p < 1.0 x 10 -15 . Data points represent individual microtubule velocities, bars indicate mean and standard deviation. f Potency of VLS-1272 at various timepoints from a global progress curve analysis (GPCA). Data presented as mean values -/+ SD from n = 11 biological replicates. g Inhibition of HCC15 cell proliferation after 4-day treatment with VLS-1272 (black) versus 1 day of VLS-1272 treatment followed by a washout and replacement in media without inhibitor for an additional 3 days (pink). Data presented as individual values from n = 2 biological replicates. h Comparison of the K I calculated by GPCA vs. the EC50 from a 2.5-day CellTiter-Glo proliferation assay in JIMT-1 cells from n = 40 compounds. Each dot represents one compound. Compounds with poor permeability are denoted by “x”. Source data are provided in the Source Data file.

Article Snippet: HCC1806 (ATCC CRL-2335) and HCC15 (DSMZ ACC-496) cells were grown in RPMI 1640 Medium (Gibco) supplemented with 10% FBS (Gibco) and 100 units/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific).

Techniques: Glo Assay, Activity Assay, Inhibition, Purification, Control, Two Tailed Test, Standard Deviation, Comparison, Proliferation Assay, Permeability

Journal: Nature Communications

Article Title: Targeting chromosomally unstable tumors with a selective KIF18A inhibitor

doi: 10.1038/s41467-024-55300-z

Figure Lengend Snippet: a Growth inhibition as measured by CellTiter-Glo of indicated cancer and non-transformed epithelial cell lines after treatment with VLS-1272 for 168 h. Data presented as individual values from n = 2 biological replicates. b Proliferation of MDA-MB-231 cells over-expressing KIF2A (CIN High ) or KIF2B (CIN Low ) treated with VLS-1272 and assessed by CellTiter-Glo after 168 h of treatment. Data presented as individual values from n = 2 biological replicates. c Growth inhibition of primary T cells isolated from healthy donors after stimulation with αCD3/CD28 beads, treated with VLS-1272 and assessed by CellTiter-Glo after 72 h. Data presented as individual values from n = 2 donors. Dead cell (propidium iodide-positive cells) enumeration by live cell imaging in the presence of vehicle or indicated VLS-1272 concentration for 150 h in JIMT-1 cells ( d ) or CAL51 cells ( e ). Data presented as mean -/+ SD from n = 3 biological replicates. f Dead cell quantification by acridine orange/propidium iodide staining following 3-day treatment with the indicated concentrations of VLS-1272 in OVCAR-3 cells. Data presented as mean -/+ SD from n = 3 biological replicates. P values are calculated compared to untreated control and labeled on the corresponding bar, using one-way ANOVA with Dunnett’s multiple comparisons test. g Western blot of cleaved Caspase 3 (MW: 17 kDa) versus full length Caspase 3 (MW: 35 kDa) after 24- and 72 h treatment of CAL51 and OVCAR-3 cells with VLS-1272. One representative experiment of 2 biological replicates is shown. Growth inhibition as measured by CellTiter-Glo of HCC15 ( h ) and HCC1806 cells ( i ) after treatment with the indicated concentration of VLS-1272 with or without 0.5 or 2 nM BAY-1217389 (MPS1 inhibitor) for 3 days. Data presented as mean values -/+ SD from n = 4 biological replicates. Source data are provided in the Source Data file.

Article Snippet: HCC1806 (ATCC CRL-2335) and HCC15 (DSMZ ACC-496) cells were grown in RPMI 1640 Medium (Gibco) supplemented with 10% FBS (Gibco) and 100 units/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific).

Techniques: Inhibition, Transformation Assay, Expressing, Isolation, Live Cell Imaging, Concentration Assay, Staining, Control, Labeling, Western Blot

Journal: Nature Communications

Article Title: Targeting chromosomally unstable tumors with a selective KIF18A inhibitor

doi: 10.1038/s41467-024-55300-z

Figure Lengend Snippet: Tumor volume measurements after administering vehicle or VLS-1272-SDD to ( a ) female SCID Beige mice bearing HCC15 tumor xenografts or ( b ) female Balb/c nude mice bearing OVCAR-3 tumor xenografts ( n = 10 mice per treatment group). Data presented as mean values -/+ SEM. p values of VLS-1272-treated groups compared to vehicle at Day 36 (HCC15) or Day 29 (OVCAR-3) are indicated and determined using one-way ANOVA with Dunnett’s multiple comparison test. Representative fluorescence images of KIF18A alone or together with α-tubulin and DAPI in tumors harvested from mice treated with vehicle or ( c ) 30 mg/kg VLS-1272 in HCC15 or ( e ) 60 mg/kg VLS-1272 in OVCAR-3. Scale bar = 10 µm ( c ) and scale bar = 5 µm ( e ). Quantification of KIF18A mislocalization measured as the ratio of KIF18A fluorescence colocalizing with α-tubulin over the amount of KIF18A colocalizing with DAPI in HCC15 ( d ) and OVCAR-3 ( f ) xenograft tumor sections. Data presented as mean values -/+ SEM from n = 5 mice per treatment group, ( d ) *** p = 0.00082 and ( f ) *** p = 0.00015 using two-tailed unpaired t test. Representative fluorescence micrographs and quantification of DAPI staining of tumors harvested from mice treated with vehicle or ( g , h ) 30 mg/kg VLS-1272 in HCC15 or ( i , j ) 60 mg/kg VLS-1272 in OVCAR-3. Scale bar = 5 µm ( g , i ). Data presented as mean values -/+ SEM from n = 5 mice per treatment group, ( h ) *** p = 0.00012 and ( j ) * p = 0.012 from two-tailed unpaired t -test. k Mitotic cell counts by pHH3 staining of tumor sections from excised OVCAR-3 xenografts. Data presented as mean -/+ SD from n = 3 mice per group. p values are indicated above the bars and determined using one-way ANOVA with Dunnett’s multiple comparison test. Source data are provided in the Source Data file.

Article Snippet: HCC1806 (ATCC CRL-2335) and HCC15 (DSMZ ACC-496) cells were grown in RPMI 1640 Medium (Gibco) supplemented with 10% FBS (Gibco) and 100 units/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific).

Techniques: Comparison, Fluorescence, Two Tailed Test, Staining

Journal: Redox biology

Article Title: A potential therapeutic strategy based on acute oxidative stress induction for wild-type NRF2/KEAP1 lung squamous cell carcinoma.

doi: 10.1016/j.redox.2024.103305

Figure Lengend Snippet: Fig. 1. High sensitivity to iROS of LUSC cell lines with wild type NFE2L2/KEAP1. (A–D) Cell death in LUSC cell lines treated with different doses of dipyridone (indicated). Cells examined included: HCC15 (exhibiting activation of the NRF2/KEAP1 pathway) (A), H226 (with normal NRF2) (B), SK-MES-1 (with altered copy number; NFE2L2>KEAP1) (C), and normal epithelial airway cells (HSAEC1/KT cells) (D). All cell lines were seeded in parallel, treated for different times with dipyridone, and maintained in exponential growth. The figure shows representative images ( × 10) of the different cells at 24h of the treatment. After 24, 48, and 72h of treatment, cells were collected and incubated with propidium iodide (PI). The percentage of PI-positive cells (dead cells) was analyzed by flow cytometry. Flow cytometry square plots show the number of PI- (alive cells) and PI+(death cells, in red) at 0.5 mg/ml of dipyridone (24h) vs untreated cells. Bar graphs show the percentage of PI+ cells per condition subtracting the background (PI+ cells in untreated cells). Percentage of cell death is expressed in relation to total cell number (100 %) (Mean ± SD, n = 3). The dashed line at 30 % cell death (maximum death in ROS treated-normal lung cells) is drawn for comparison. ***P > 0.001 unpaired t-test; n.s. non-statistically significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The following LUSC cell lines were purchased from the American Type Culture Collection [SK-MES-1 (HTB-58), H520 (HTB182), and H226 (CRL-5826)] and one from the DSMZ collection [HCC-15 (ACC- 496)].

Techniques: Activation Assay, Incubation, Flow Cytometry, Comparison

Journal: Redox biology

Article Title: A potential therapeutic strategy based on acute oxidative stress induction for wild-type NRF2/KEAP1 lung squamous cell carcinoma.

doi: 10.1016/j.redox.2024.103305

Figure Lengend Snippet: Fig. 2. Different iROS inducers trigger LUSC cell death. (A) mRNA expression of NFE2L2 and its target gene NQO1 determined in H226 and HCC15 cells treated with dipyridone at different concentrations and times (indicated). The initial cycle (indicated as C) at which the genes are detected in untreated cells is indicated. Graph bars represents the Mean±SD of the RQ levels referred to gene expression in control cells, n = 3. (B) NRF2 and NQO1 protein expression levels in cells treated as in (A) were tested in WB. Graph bars (at the bottom) represent NRF2 and NQO1 signals (mean ± SD, n = 2) corrected by the loading control (α tubulin) and referred to the levels at 24h of dipyridone treatment (100 %). (C, D) Representative images of H226 and HCC15 cells ( × 10) treated with vehicle, oxPAPC, or dipyridone (positive control) for 24h (C); or of H226 and HCC15 cells treated with anisomycin for 24h (D). Graphs on the right show the percentage of PI+ cells in untreated vs treated cells (indicated) as determined by flow cytometry. Graph bars represent the Mean ± SD, n = 3(C, D). Statistical significance was determined by one-way ANOVA with Dunett test as a post hoc analysis; (*) P < 0.05, (**) P < 0.01, (***) P < 0.001 or using Unpaired t-test: *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The following LUSC cell lines were purchased from the American Type Culture Collection [SK-MES-1 (HTB-58), H520 (HTB182), and H226 (CRL-5826)] and one from the DSMZ collection [HCC-15 (ACC- 496)].

Techniques: Expressing, Gene Expression, Control, Positive Control, Flow Cytometry

Journal: Redox biology

Article Title: A potential therapeutic strategy based on acute oxidative stress induction for wild-type NRF2/KEAP1 lung squamous cell carcinoma.

doi: 10.1016/j.redox.2024.103305

Figure Lengend Snippet: Fig. 6. SETD5 silencing induces G6PD de-repression and triggers cell death in normal-NRF2 LUSC cells. (A) Comparison of G6PD, TKT, and TALDO levels (RQ values vs GAPDH and TBP) in control and dipyridone-treated (0.5 mg/ml; 24 or 48 h) H226 or HCC15 cells. Graphs show the mRNA levels of the genes (as RQ values) for the different conditions. The cycle at which G6PD, TKT, and TALDO appear in untreated cells is indicated and shows higher levels that do not change with dipyridone in active-NFR2 cells. (B) H226 or HCC15 cells were activated with dipyridone (0.5 mg/ml; 24 and 48h). Lysates (50 μg) were examined in WB (indicated). Graph represent G6PD WB signal at 48h, corrected with the loading control and referred to maximal (100 %). (C) 4×105 H226 or HCC15 cells were seeded in p60 plates 24h prior to transfection with control or SETD5 specific siRNA (72 h). Cells were collected for RT-qPCR. Graphs as in (A). (D) 105 H226 or HCC15 cells were seeded in 12-well plates 24 h prior to addition of vehicle, dipyridone (0.75 mg/ml), or AG1 (indicated). Representative flow cytometry images of the cells after 75 μM AG1 treatment (24 h); on the right, percentage of death. *P < 0.05; **P < 0.01; ***P < 0.001 Student’s t-test; (*) P < 0.05, (**) P < 0.01, (***) P < 0.001; two-way ANOVA with Bonferroni post-test.

Article Snippet: The following LUSC cell lines were purchased from the American Type Culture Collection [SK-MES-1 (HTB-58), H520 (HTB182), and H226 (CRL-5826)] and one from the DSMZ collection [HCC-15 (ACC- 496)].

Techniques: Comparison, Control, Transfection, Quantitative RT-PCR, Flow Cytometry

Journal: Redox biology

Article Title: A potential therapeutic strategy based on acute oxidative stress induction for wild-type NRF2/KEAP1 lung squamous cell carcinoma.

doi: 10.1016/j.redox.2024.103305

Figure Lengend Snippet: Fig. 7. Transient iROS slows WT NFE2L2/KEAP1 LUSC organoid growth. (A) 104 H226 or HCC15 cells were seeded per well in 96-well ultralow attachment plates. After 96 h, medium was renovated or replaced with media plus dipyridone (0.75 or 1 mg/ml). 24h later, DRAQ7 probe was added (30 min). Representative images ( × 10) of confocal microscopy. Graphs (mean ± SD) represent the cell-death fold change (n ≥4) compared to untreated spheroids at different times. (B) mRNA levels of PIK3R2 and SETD5 in a battery of LUSC PDX (patient derived xenografts). Graphs show the RQ values (versus GAPDH) relative to gene expression levels in HCC15 control. Squared samples represent high RQs; dashed squared those with low RQs. (C) Extracts obtained from PDX (50 μg) were examined in WB with indicated antibodies (n > 20 tumors examined). The graphs show the percent of signal referred to maximal p85β signal (TP3 signal, 100 %) or, in the case of SETD5, to SETD5 levels in tumor T34 (100 %). These blots were performed twice with similar results. (D) mRNA levels of NFE2L2, AKR1C3, and NOQ1 in a collection of LUSC PDX. Graphs as in (B). PDX expressing NRF2 effectors constitutively (squared high RQs; e.g.TP13) or normal-NRF2 organoids with low constitutive expression of NRF2 targets (dashed squared, low RQs; e.g.TP34) but high levels of SETD5 and PIK3R2 mRNA (TP34) were chosen. (E) Cells from the two selected LUSC PDX- derived organoids were seeded (40,000 cells/well) in confocal plates, after 24h, culture media were replaced with media or media containing dipyridone (0.75; 1 mg/ml) (24h). DRAQ7 probe was added (30 min). Representative images from confocal microscopy. Graphs represent the number of DRAQ7+ dead cells per field (mean ± SD) in each organoid (n ≥4). *P < 0.05; **P < 0.01; ***P < 0.001 Unpaired t-test.

Article Snippet: The following LUSC cell lines were purchased from the American Type Culture Collection [SK-MES-1 (HTB-58), H520 (HTB182), and H226 (CRL-5826)] and one from the DSMZ collection [HCC-15 (ACC- 496)].

Techniques: Confocal Microscopy, Battery, Derivative Assay, Gene Expression, Control, Expressing

Journal: Redox biology

Article Title: A potential therapeutic strategy based on acute oxidative stress induction for wild-type NRF2/KEAP1 lung squamous cell carcinoma.

doi: 10.1016/j.redox.2024.103305

Figure Lengend Snippet: Fig. 8. Transient iROS slows WT NFE2L2/KEAP1 LUSC tumor growth in vivo. (A) H226 cells were expanded in culture and grafted subcutaneously into nude mice (107 cells in 100 μl of PBS plus 100 μl matrigel). The graph illustrates the mean tumor volume in mm3 ± SD, n = 12) measured at different times after initiation of dipyridone (100 mg/kg) treatment. (B) Representative images of control and treated mice harboring LUSC tumors and of a sample of these tumors. (C) Different gene mRNA expression levels analyzed in control and dipyridone-treated LUSC tumors. Significant decreases were found in miR-126 and SETD5 levels. (D) Cell lysates (50 μg) from untreated (control, Ctr) and treated tumors (from A) were examined in WB. The graph shows the signal intensity in the control and treated tumors (normalized to loading control) and referred to the mean p85βPI3K content in untreated tumors (100 %). MW, Molecular weight. (***) P < 0.001 (A) two-way ANOVA test followed by Tukey posttest; (C, D) *P < 0.05; **P < 0.01; ***P < 0.001 Unpaired t-test.

Article Snippet: The following LUSC cell lines were purchased from the American Type Culture Collection [SK-MES-1 (HTB-58), H520 (HTB182), and H226 (CRL-5826)] and one from the DSMZ collection [HCC-15 (ACC- 496)].

Techniques: In Vivo, Control, Expressing, Molecular Weight