vls-1272  (Amgen)

Journal: Nature Communications

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

doi: 10.1038/s41467-024-55300-z

Figure Lengend Snippet: DEPMAP gene essentiality data was used to identify genes required for proliferation of CIN High cells. a Categorization of CIN across 1660 cell lines as CIN High (top quartile), CIN Med or CIN Low (bottom quartile) as measured by fraction genome altered (FGA). Data presented as mean values. Each dot represents 1 cell line. b Volcano plot visualizing the odds ratio from a Fisher’s Exact test to determine the association between RNAi essentiality scores and CIN, which identified genes that are significantly essential in CIN High cell lines (FDR < 0.25). Each dot represents one gene. Genes in green are more essential in CIN High cells, genes in red are more essential in CIN Low cells, and genes in blue did not show differential essentiality. KIF18A p value = 0.0072 with a Fisher’s Exact test. c Cell lines requiring KIF18A for proliferation (Ess) exhibit higher median FGA compared to cell lines not requiring KIF18A for proliferation (Non-Ess) based on RNAi essentiality scores across 748 cell lines. Data presented as median (center) with 25th to 75th percentile bounds (boxes), minimum and maximum (whiskers). ** p = 0.006 by unpaired, 2-tailed t test. Source data are provided in the Source Data file.

Article Snippet: For side-by-side comparison of VLS-1272 and AM-1882, reactions were performed with 1.25 nM KIF18A and 50 uM ATP for 30 min. AM-1882 was purchased from MedChemExpress (KIF18A-IN-1; Cat. No.: HY-145034).

Techniques:

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 Micronuclei levels (ratio of total micronuclei to total primary nuclei) in MDA-MB-231 cells over-expressing KIF2B or KIF2A compared to the parental line. Data presented as mean values -/+ SD from n = 6 biological replicates, *** p = 1.01 x 10 − 4 using one-way ANOVA with Dunnett’s multiple comparisons test. b Defective anaphases (as percentage of total anaphases) in MDA-MB-231 cells overexpressing KIF2A or KIF2B. Data presented as mean values from n = 2 biological replicates. c Western blot confirming CRISPR/Cas9-mediated KIF18A knockout. One representative experiment of 2 biological replicates is shown. d Proliferation (luminescence measured by CellTiter-Glo assay) upon KIF18A knockout. Data presented as individual values from n = 2 biological replicates. (e) Western blot of KIF18A knockdown (K1 and K2) compared to control siRNA (C1 and C2) 72 h after replating. Top panel: arrow indicates KIF18A (the highest band), bottom band is non-specific. One representative experiment of 2 biological replicates is shown. f Viability of cells shown in (e) 72 h after replating following siRNA transfection. Computed CIN values (FGA) of cell lines are indicated below the name. Data are presented as mean values from 2 biological replicates. g Western blot of KIF18A expression in engineered Dox-inducible KIF18A (shRNA 1 and 2) and Control (non-targeting) shRNA in JIMT-1 cells after 7 days Dox treatment. One representative experiment of 2 biological replicates is shown. h Proliferation of engineered Dox-inducible JIMT-1 cells measured after 10 days Dox treatment. Data presented as mean values +/- SD from n = 3 biological replicates. p values are indicated above the bars and determined by 2-way ANOVA with Tukey’s multiple comparisons test. i Cell cycle population frequencies in Dox-inducible KIF18A and Control shRNA JIMT-1 cells after 72 h of Dox treatment. Data presented as mean values from n = 2 biological replicates. j Tumor volume of Dox-inducible shRNA JIMT-1 xenografts implanted in SCID Beige mice ( n = 4 mice per group). Data presented as mean values -/+ SEM. k Western blot of KIF18A expression from JIMT-1 xenograft tumors excised 4 days after initiation of Dox treatments. Source data are provided in the Source Data file.

Article Snippet: For side-by-side comparison of VLS-1272 and AM-1882, reactions were performed with 1.25 nM KIF18A and 50 uM ATP for 30 min. AM-1882 was purchased from MedChemExpress (KIF18A-IN-1; Cat. No.: HY-145034).

Techniques: Expressing, Western Blot, CRISPR, Knock-Out, Glo Assay, Knockdown, Control, Transfection, shRNA

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: For side-by-side comparison of VLS-1272 and AM-1882, reactions were performed with 1.25 nM KIF18A and 50 uM ATP for 30 min. AM-1882 was purchased from MedChemExpress (KIF18A-IN-1; Cat. No.: HY-145034).

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: Selectivity summary for VLS-1272 against a panel of human and mouse kinesins

Article Snippet: For side-by-side comparison of VLS-1272 and AM-1882, reactions were performed with 1.25 nM KIF18A and 50 uM ATP for 30 min. AM-1882 was purchased from MedChemExpress (KIF18A-IN-1; Cat. No.: HY-145034).

Techniques:

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 Representative immunofluorescence images of HCC1806 cells treated with DMSO (top) or 37.5 nM VLS-1272 (bottom). Scale bar = 5 µm. One representative image from the treatment and control group from two biological replicates is shown. b Quantification of KIF18A mis-localization in mitotic HCC1806 cells measured as the ratio of KIF18A colocalizing with the α-tubulin spindle to KIF18A colocalizing with DNA (DAPI). Colocalization masks were generated in Harmony high-content analysis software. Data presented as violin plots with the mean (solid line) and quartiles (dashed lines) from n = 233-803 mitotic cells. Adjusted p value is calculated compared to untreated control and labeled next to each condition, using one-way ANOVA with Bonferroni’s multiple comparisons test. c Representative fluorescent images of DAPI-stained mitotic DNA in HCC1806 cells treated with DMSO or VLS-1272. Scale bar = 5 µm. One representative image from the treatment and control group from two biological replicates is shown. d Quantification of the area of DAPI staining (µm 2 ) in pHH3-positive cells treated with increasing doses of VLS-1272. The area of the DAPI-stained DNA was calculated in Harmony high-content analysis software. Data presented as violin plots with the mean (solid line) and quartiles (dashed lines) from n = 652–1434 mitotic cells. Adjusted p value is calculated compared to untreated control and labeled next to each condition, using one-way ANOVA with Bonferroni’s multiple comparisons test. e Quantification of mitotic HCC1806 cells identified by positive staining for pHH3 compared to the number of total DAPI-stained nuclei. The mitotic cell population was calculated in Harmony high-content analysis software. f Percentage of total micronuclei to primary nuclei in cells treated with VLS-1272, calculated in Harmony high-content analysis software. e , f Data presented as mean values with individual data points shown from n = 2 biological replicates. Source data are provided in the Source Data file.

Article Snippet: For side-by-side comparison of VLS-1272 and AM-1882, reactions were performed with 1.25 nM KIF18A and 50 uM ATP for 30 min. AM-1882 was purchased from MedChemExpress (KIF18A-IN-1; Cat. No.: HY-145034).

Techniques: Immunofluorescence, Control, Generated, High Content Screening, Software, Labeling, Staining

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: For side-by-side comparison of VLS-1272 and AM-1882, reactions were performed with 1.25 nM KIF18A and 50 uM ATP for 30 min. AM-1882 was purchased from MedChemExpress (KIF18A-IN-1; Cat. No.: HY-145034).

Techniques: Comparison, Fluorescence, Two Tailed Test, Staining

Journal: Nature communications

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

doi: 10.1038/s41467-024-55300-z

Figure Lengend Snippet: Fig. 3 | Development of KIF18A inhibitors. 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

Article Snippet: For single dose PK studies, 6–8-week-old male CD-1 mice (Jihui Laboratory Animal Co. Ltd.) were administered 1mg/kg VLS-1272 intravenously into the tail vein, or 30mg/kg VLS1272-SDD (vehicle: 5% TPGS (Tocofersolan (MCE cat #109605)) and 0.5%Methylcellulose (400cp) (Aladdin, cat #K2005343) in ddH2O) via oral gavage.

Techniques: Glo Assay, Activity Assay, Inhibition, Control