Journal: eBioMedicine

Article Title: Customised design of antisense oligonucleotides targeting EGFR driver mutants for personalised treatment of non-small cell lung cancer

doi: 10.1016/j.ebiom.2024.105356

Figure Lengend Snippet: RBCEVs loaded with EGFR mutant-specific ASOs exhibit superior anti-cancer effect compared to TKIs. ( a ) Flow cytometric analysis of Annexin V/SYTOX Blue staining in H1975 cells 24 h post-treatment with 200 nM of Icotinib, Afatinib, Osimertinib, unloaded RBCEVs (EVs) or ASO-loaded RBCEVs (L858R ASO-EVs, T790M ASO-EVs). ( b ) Proportion of early apoptotic (Annexin + , SYTOX Blue − ), necrotic (Annexin − , SYTOX Blue + ), and late apoptotic (Annexin + , SYTOX Blue + ) H1975 cells treated as described in (A) (n = 3). ( c ) Relative cell counts of H1975 cells two days after treatment with Icotinib, Afatinib, Osimertinib, unloaded RBCEVs or ASO-loaded RBCEVs at equal concentrations as shown in (a), determined by the CCK8 assay (n = 3 biological repeats). The graphs present mean ± SEM. ∗∗∗P < 0.001, determined using One-Way ANOVA test.

Article Snippet: To assess the anti-tumour effect of ASOs delivered by RBCEVs, H1975 cells were seeded in a 96-well plate at a density of 1.0 x 10 4 cells per well, prior to incubation with TKIs (HaoYuan ChemExpress, China), or RBCEVs loaded with either EGFR L858R ASO or EGFR T790M ASO at equivalent molar doses of ASO and TKIs.

Techniques: Mutagenesis, Staining, CCK-8 Assay

Journal: Cancer research

Article Title: Insights into ALK-driven cancers revealed through development of novel ALK tyrosine kinase inhibitors

doi: 10.1158/0008-5472.CAN-10-3879

Figure Lengend Snippet: In vitro potency and selectivity of ALK TKIs The degree of inhibition from Kinome scan is shown for each protein.

Article Snippet: We next performed KINOME scan (Ambit Biosciences) analysis to test the specificity of the ALK TKIs.

Techniques: In Vitro, Inhibition

Journal: Cancer research

Article Title: Insights into ALK-driven cancers revealed through development of novel ALK tyrosine kinase inhibitors

doi: 10.1158/0008-5472.CAN-10-3879

Figure Lengend Snippet: (A-D) H3122 lung cancer cells containing the EML4-ALK E13;A20 fusion, H2228 lung cancer cells harboring the EML4-ALK E6a/b;A20 fusion, SUDHL-1 lymphoma cells containing NPM-ALK fusion, and SY5Y neuroblastoma cells with an activating mutation within the ALK kinase domain (ALK F1174L) were treated with ALK TKIs or vehicle for 72h. Cell titer blue assays were performed to assess growth inhibition. Each point represents hextuplicate replicates. Data are presented as the percentage of viable cells compared to control (vehicle only treated) cells. See methods for details. (E) Apoptosis is induced by X-376 treatment. H3122 cells were treated with increasing concentrations of X-376 for 72h. Cells were stained with annexin V (AV) and propidium iodide (PI) and counted on a FACSCanto II machine. Viable cells are defined as the AV/PI double negative population. Apoptotic cells are defined as the sum of AV positive, PI negative plus AV/PI double positive cell populations. (F) 293 cells were transiently transfected with 3Flag-EML4-ALK E13;A20. At 48 hours post transfection, the cells were treated with increasing amounts of the ALK TKIs for 2 hours. Lysates were subjected to immunoblotting with antibodies specific for the indicated proteins. (G) H3122 lung cancer cells containing the EML4-ALK E13;A20 fusion were treated with increasing amounts of ALK TKIs for 1 hour as indicated. Lysates were subjected to immunoblotting with the specified antibodies. The asterisks (*) in the ALK and phospho-AKT blot indicate an empty lane (no lysate loaded) on the gel.

Article Snippet: We next performed KINOME scan (Ambit Biosciences) analysis to test the specificity of the ALK TKIs.

Techniques: Mutagenesis, Inhibition, Staining, Transfection, Western Blot

Journal: Cancer research

Article Title: Insights into ALK-driven cancers revealed through development of novel ALK tyrosine kinase inhibitors

doi: 10.1158/0008-5472.CAN-10-3879

Figure Lengend Snippet: IC 50 values for PF-1066, X-376, and X-396 in a panel of cancer cell lines Cell lines were treated with ALK TKIs for 72h. Cell titer blue assays were performed to assess cell proliferation. IC 50 values were calculated using GraphPad Prism software with a non-linear regression. Each experiment was performed at least 2 times. NT: not tested.

Article Snippet: We next performed KINOME scan (Ambit Biosciences) analysis to test the specificity of the ALK TKIs.

Techniques: Software

Journal: Cancer research

Article Title: Insights into ALK-driven cancers revealed through development of novel ALK tyrosine kinase inhibitors

doi: 10.1158/0008-5472.CAN-10-3879

Figure Lengend Snippet: (A) 293 cells were transiently transfected with expression plasmids encoding various ALK fusions. At 48 hours post transfection, the cells were treated with increasing amounts of X-376 for 2 hours. Lysates were subjected to immunoblotting with antibodies specific for the indicated proteins. (B-C) 293 cells were transiently transfected with 3Flag-EML4-ALK E13;A20 WT alongside 3Flag-EML4-ALK E13;A20 L1196M (B) or 3Flag-EML4-ALK E13;A20 C1156Y (C). At 48 hours post transfection, the cells were treated with increasing amounts of the indicated ALK TKI for 2 hours. Lysates were subjected to immunoblotting with antibodies specific for the indicated proteins. The exposure of the pALK blot was selected to highlight the difference in baseline phosphorylation between wild-type and mutants. (D) Western blot demonstrating EML4-ALK E13;A20 WT, L1196M, and C1156Y expression in Ba/F3 cell lines. (E) Ba/F3 cells expressing EML4-ALK E13;A20 WT, L1196M, or C1156Y were treated with ALK TKIs or vehicle for 72h. Cell titer blue assays were performed to assess growth inhibition. Each point represents hextuplicate replicates. Data are presented as the percentage of viable cells compared to control (vehicle only treated) cells. See methods for details.

Article Snippet: We next performed KINOME scan (Ambit Biosciences) analysis to test the specificity of the ALK TKIs.

Techniques: Transfection, Expressing, Western Blot, Inhibition