human kras mutant colorectal cancer cell lines hct116  (ATCC)

Journal: Journal of Clinical Oncology

Article Title: Onvansertib in Combination With Chemotherapy and Bevacizumab in Second-Line Treatment of KRAS -Mutant Metastatic Colorectal Cancer: A Single-Arm, Phase II Trial

doi: 10.1200/JCO-24-01266

Figure Lengend Snippet: PLK1 regulates the hypoxia pathway, related to . (A, B) KRAS -mutant CRC cells were treated with DMSO or Onv at the indicated doses for 20 hours and then exposed to hypoxia or kept in normoxia for 4 hours. (A) Heatmap of hypoxia-related genes significantly regulated by Onv in HCT116 and SW620 cells based on the RNA-seq analysis. (B) Expression of hypoxia-related genes in LoVo and DLD-1 cells, assessed by RT-qPCR and normalized to the housekeeping gene RPLP0 . Bar graphs represent expression relative to Normoxia_DMSO sample. (C, D) SW620 and HCT116 cells were transfected with nontargeting control siRNA (siNTC) or PLK1 targeting siRNA (siPLK1) for 20 hours and then exposed to hypoxia for 4 hours. (C) Left: Simple Western images of PLK1, HIF1α, and β-actin. Right: HIF1α and PLK1 protein expression normalized to β-actin. (D) Expression of hypoxia-related genes assessed by RT-qPCR and normalized to the housekeeping gene RPLP0 . Bar graphs represent expression relative to Normoxia_siNTC. (B-D) Data are shown as mean ± SEM of at least three independent biological replicates. HIF1α, hypoxia-inducible factor 1α; Hx, hypoxia; Nx, normoxia; Onv, onvansertib; PLK1, polo-like kinase 1; RT-qPCR, real-time quantitative polymerase chain reaction.

Article Snippet: Human KRAS -mutant colorectal cancer cell lines HCT116, SW620, LoVo, and DLD-1 (ATCC, Manassas, VA) were cultured in RPMI (Cat# 30-2001, ATCC) supplemented with 10% FBS (Cat#16000069, ThermoFisher Scientific, Waltham, MA) and 1× penicillin-streptomycin solution (Cat#30-2300, ATCC).

Techniques: Mutagenesis, RNA Sequencing, Expressing, Quantitative RT-PCR, Transfection, Control, Simple Western, Real-time Polymerase Chain Reaction

Journal: ACS Chemical Biology

Article Title: Distal Covalent Targeting Suppresses Signaling of Oncogenic K‑Ras(G13C) in Cancer Cells

doi: 10.1021/acschembio.5c00249

Figure Lengend Snippet: (A) Alignment of K-Ras­(G13C) to adagrasib-bound K-Ras­(G12C). (B) Modular design of shift-register electrophiles to repurpose K-Ras­(G12C) covalent inhibitors for the G13C mutation. (C) Proximity scanning of Cys13 accessibility using unbranched alkyl linkers. (D) Labeling efficiency of G13C-targeting covalent inhibitors with various linker lengths. Conditions: K-Ras­(G13C)•GDP or •GppNHp (1 μM), compound (100 μM), RT incubation. Each box represents the mean of two independent runs. (E) Schematic summary of an expanded linker screening campaign. (F) Lead 1,3-disubstituted aromatic linked compounds and their covalent modification kinetics. Conditions: K-Ras­(G13C)•GDP or •GppNHp (200 nM), compound (10 μM), RT incubation.

Article Snippet: HCT116/KRAS G13C heterozygous and homozygous (SL753) cancer biomarker mutant cell lines were obtained from GeneCopoeia, Inc. (Rockville, MD).

Techniques: Mutagenesis, Labeling, Incubation, Modification

Journal: ACS Chemical Biology

Article Title: Distal Covalent Targeting Suppresses Signaling of Oncogenic K‑Ras(G13C) in Cancer Cells

doi: 10.1021/acschembio.5c00249

Figure Lengend Snippet: (A) Chemical structure of lead compound G13Ci-22 . (B) Aligned ligand poses of G13Ci-22 (CovDock) and MRTX1133 (PDB Entry 7RPZ ) in the corresponding Switch-II Pocket. (C) Covalent modification kinetics of K-Ras­(G13C) by inhibitors with the optimal 3-acrylamidobenzene carbonyl linker and alternative Switch-II Pocket binding moieties. Conditions: K-Ras­(G13C)•GDP or •GppNHp (200 nM), Compound (10 μL), RT incubation. (D) Chemical structures of the alternative Switch-II Pocket binding moieties. The optimal linker and Cys-warhead, same with those in G13Ci-22 , were omitted in the chemical structures.

Article Snippet: HCT116/KRAS G13C heterozygous and homozygous (SL753) cancer biomarker mutant cell lines were obtained from GeneCopoeia, Inc. (Rockville, MD).

Techniques: Modification, Binding Assay, Incubation

Journal: ACS Chemical Biology

Article Title: Distal Covalent Targeting Suppresses Signaling of Oncogenic K‑Ras(G13C) in Cancer Cells

doi: 10.1021/acschembio.5c00249

Figure Lengend Snippet: (A) Mutant selectivity of G13Ci-22 against relevant cyslight K-Ras proteins. (B) Thermal stabilization of K-Ras­(G13C) proteins due to covalent modification by G13Ci-22 . (C) Schematic description of guanine nucleotide cycles of K-Ras, K-Ras­(G13C), and covalently inhibited K-Ras­(G13C) GTPases. G13Ci-22 inhibited Ras-RafRBD binding (D), GTPase activity (E), and nucleotide exchange (F) of K-Ras­(G13C) oncoprotein.

Article Snippet: HCT116/KRAS G13C heterozygous and homozygous (SL753) cancer biomarker mutant cell lines were obtained from GeneCopoeia, Inc. (Rockville, MD).

Techniques: Mutagenesis, Modification, Binding Assay, Activity Assay

Journal: ACS Chemical Biology

Article Title: Distal Covalent Targeting Suppresses Signaling of Oncogenic K‑Ras(G13C) in Cancer Cells

doi: 10.1021/acschembio.5c00249

Figure Lengend Snippet: (A) Immunoblot of HCT116 cell and its genetically engineered derivative cells treated with G13Ci-22 at various concentrations. (B) Time-course immunoblot of the homozygous HCT116 (K-Ras­(G13C/G13C)) cell treated with 10 nM of G13Ci-22 . (C) Immunoblot of human cancer cell lines NCI-H1355 and NCI-H1734, which harbor K-Ras­(G13C) mutation, treated with G13Ci-22 .

Article Snippet: HCT116/KRAS G13C heterozygous and homozygous (SL753) cancer biomarker mutant cell lines were obtained from GeneCopoeia, Inc. (Rockville, MD).

Techniques: Western Blot, Mutagenesis

Journal: Chemmedchem

Article Title: Sequence‐Selective Covalent CaaX‐Box Receptors Prevent Farnesylation of Oncogenic Ras Proteins and Impact MAPK/PI3 K Signaling

doi: 10.1002/cmdc.202100167

Figure Lengend Snippet: Thiol‐based CaaX‐box inhibitors reduce tumor cell viability. A ) Dose response curves of HCT116 after 72 h incubation with MFZ‐112 ( 20 , no sigmoidal curve) MFZ‐115 ( 23 , IC 50 =538±58 μM), MFZ‐117 ( 25 , IC 50 =445±50 μM) and MFZ‐148 ( 27 , IC 50 =413±10 μM); Dose response curves of NCI−H358 after 72 h incubation with MFZ‐112 ( 20 , no sigmoidal curve fit), MFZ‐115 ( 23 , IC 50 =1,741±0,39 mM), MFZ‐117 ( 25 , IC 50 =488±50 μM) and MFZ‐148 ( 27 , IC 50 =312±36 μM). B , C ) Western Blot analysis of RAS‐dependent Erk phosphorylation of serum starved HCT116 cells after 2 h and 20 h incubation with potential CaaX‐box inhibitors. Data is represented as mean ± SD, n=3.

Article Snippet: KRAS mutant human HCT116 colon cancer cells and NCI−H358 NSCLC cells were maintained from the American Type Culture Collection (ATCC).

Techniques: Incubation, Western Blot, Phospho-proteomics

Journal: Chemmedchem

Article Title: Sequence‐Selective Covalent CaaX‐Box Receptors Prevent Farnesylation of Oncogenic Ras Proteins and Impact MAPK/PI3 K Signaling

doi: 10.1002/cmdc.202100167

Figure Lengend Snippet: CaaX‐box inhibitors modify RAS‐dependent signaling cascades. A ) Treatment of NCI−H358 cells with thiol‐based inhibitors MFZ‐115 ( 23 ) and MFZ‐117 ( 25 ) for 20 h, followed by 10 min. stimulation von 100 ng/ml EGF under serum‐free conditions. B ) Western blot analysis of NCI−H358 and HCT116 cells after 20 h treatment with 4‐fluoroproline derivate MFZ‐148 ( 27 ). n=3.

Article Snippet: KRAS mutant human HCT116 colon cancer cells and NCI−H358 NSCLC cells were maintained from the American Type Culture Collection (ATCC).

Techniques: Western Blot