Journal: bioRxiv

Article Title: Preclinical Prediction of Resistance and Optimization of Sequential Therapy for ALK-positive Lung Cancer Using Next-generation ALK Inhibitors

doi: 10.1101/2025.05.26.656085

Figure Lengend Snippet: The sensitivity of alectinib-resistant mutants against zotizalkib, gilteritinib, and neladalkib. a Chemical structure of zotizalkib. b Chemical structure of gilteritinib. c Chemical structure of neladalkib. d–f Sensitivity evaluation of alectinib-resistant mutants against zotizalkib ( d ), gilteritinib ( e ), and neladalkib ( f ). Ba/F3 cells expressing EML4–ALK variant 1 (v1) plus G1202R or I1171N were exposed to each inhibitor for 72 h. Cell viability was evaluated by CCK-8, with absorbance measured at 450 nm. g–i Immunoblotting evaluation of the suppression of phosphorylated ALK expression in alectinib-resistant mutants by zotizalkib ( g ), gilteritinib ( h ), or neladalkib ( i ). Ba/F3 cells expressing EML4–ALK v1 and each resistance mutation were treated with different inhibitors for 3 h. Next, immunoblotting was used to detect the indicated protein in cell lysates. CCK-8 Cell Counting Kit-8, EML4 echinoderm microtubule-associated protein-like 4, ALK anaplastic lymphoma kinase

Article Snippet: Subsequently, the membranes were incubated overnight at 4℃ with gentle agitation in primary antibody dilution buffer (phosphorylated ALK [Y1604; Cell Signaling Technologies, Danvers, MA, USA; #3341, 1:1000], ALK [Cell Signaling Technologies; #3791, 1:2000], and α-tubulin [FUJIFILM Wako; #013-25033, 1:4000]).

Techniques: Expressing, Variant Assay, CCK-8 Assay, Western Blot, Mutagenesis, Cell Counting

Journal: bioRxiv

Article Title: Preclinical Prediction of Resistance and Optimization of Sequential Therapy for ALK-positive Lung Cancer Using Next-generation ALK Inhibitors

doi: 10.1101/2025.05.26.656085

Figure Lengend Snippet: Zotizalkib-resistant mutations emerging from G1202R-positive ALK and their cross-sensitivity to other ALK-TKIs. a Predicted zotizalkib-resistant mutations (n = 54). Mutant libraries of Ba/F3 cells were exposed to zotizalkib (100 nM) for 2 weeks. b Sensitivity evaluation of predicted zotizalkib-resistant mutants against zotizalkib. Ba/F3 cells expressing EML4–ALK variant 1 and a zotizalkib-resistant mutation were exposed to zotizalkib for 72 h. Cell viability was evaluated using the CCK-8 assay, with absorbance measured at 450 nm. c Immunoblotting evaluation of the suppression of phosphorylated ALK expression in predicted zotizalkib-resistant mutants by zotizalkib. Ba/F3 cells expressing EML4–ALK variant 1 and a resistance mutation were treated with zotizalkib for 3 h. Next, immunoblotting was used to detect the indicated protein in cell lysates. d–g Sensitivity evaluation of predicted zotizalkib-resistant mutants against alectinib ( d ), crizotinib ( e ), lorlatinib ( f ), and brigatinib ( g ). Ba/F3 cells expressing EML4–ALK variant 1 with each resistance mutation were exposed to zotizalkib for 72 h. Cell viability was evaluated by CCK-8 and absorbance at a wavelength of 450 nm. CCK-8 Cell Counting Kit-8, EML4 echinoderm microtubule-associated protein-like 4, ALK anaplastic lymphoma kinase, TKIs tyrosine kinase inhibitors

Article Snippet: Subsequently, the membranes were incubated overnight at 4℃ with gentle agitation in primary antibody dilution buffer (phosphorylated ALK [Y1604; Cell Signaling Technologies, Danvers, MA, USA; #3341, 1:1000], ALK [Cell Signaling Technologies; #3791, 1:2000], and α-tubulin [FUJIFILM Wako; #013-25033, 1:4000]).

Techniques: Mutagenesis, Expressing, Variant Assay, CCK-8 Assay, Western Blot, Cell Counting

Journal: bioRxiv

Article Title: Preclinical Prediction of Resistance and Optimization of Sequential Therapy for ALK-positive Lung Cancer Using Next-generation ALK Inhibitors

doi: 10.1101/2025.05.26.656085

Figure Lengend Snippet: Gilteritinib-resistant mutations emerging from I1171N-positive ALK and their cross-sensitivity to other ALK-TKIs. a Predicted gilteritinib-resistant mutations (n = 49). Mutant libraries of Ba/F3 cells were exposed to gilteritinib (100 nM) for 2 weeks. b Sensitivity of the predicted resistant mutants to gilteritinib. Ba/F3 cells expressing EML4–ALK variant 1 and each mutation were exposed to gilteritinib for 72 h. Cell viability was evaluated by the CCK-8 assay, with absorbance measured at 450 nm. c Immunoblotting evaluation of the suppression of phosphorylated ALK expression in each resistant mutant by gilteritinib. Gilteritinib was given to Ba/F3 cells expressing EML4-ALK variant 1 with each resistance mutation for 3 h. Next, immunoblotting was used to detect the indicated protein in cell lysates. d–g Sensitivity evaluation of each resistance mutants against alectinib ( d ), crizotinib ( e ), lorlatinib ( f ), and brigatinib ( g ). Ba/F3 cells expressing EML4-ALK variant 1 with each resistance mutation were exposed to zotizalkib for 72 h. Cell viability was evaluated by the CCK-8 assay, with absorbance measured at 450 nm. CCK-8 Cell Counting Kit-8, EML4 echinoderm microtubule-associated protein-like 4, ALK anaplastic lymphoma kinase, TKIs tyrosine kinase inhibitors

Article Snippet: Subsequently, the membranes were incubated overnight at 4℃ with gentle agitation in primary antibody dilution buffer (phosphorylated ALK [Y1604; Cell Signaling Technologies, Danvers, MA, USA; #3341, 1:1000], ALK [Cell Signaling Technologies; #3791, 1:2000], and α-tubulin [FUJIFILM Wako; #013-25033, 1:4000]).

Techniques: Mutagenesis, Expressing, Variant Assay, CCK-8 Assay, Western Blot, Cell Counting

Journal: bioRxiv

Article Title: Preclinical Prediction of Resistance and Optimization of Sequential Therapy for ALK-positive Lung Cancer Using Next-generation ALK Inhibitors

doi: 10.1101/2025.05.26.656085

Figure Lengend Snippet: Neladalkib-resistant mutations emerging from G1202R- or I1171N-positive ALK and their cross-sensitivity to other ALK-TKIs. a Predicted neladalkib-resistant mutations with G1202R (n = 7). Mutant libraries of Ba/F3 cells were exposed to neladalkib (50 nM) for 2 weeks. b Sensitivity evaluation of the G1202R + L1196M compound variant against neladalkib. Ba/F3 cells expressing EML4–ALK variant 1 and different mutants were exposed to neladalkib for 72 h. Cell viability was evaluated by the CCK-8, with absorbance measured at 450 nm. c Predicted neladalkib-resistant mutations with I1171N (n = 64). Mutant libraries of Ba/F3 cells were exposed to neladalkib (500 nM) for 2 weeks. Other mutations included the triple mutation I1171N + C1156Y + D1203N, as well as I1171N + L1196M and I1171N + V1130L, both of which remained sensitive to neladalkib. d Sensitivity of predicted mutations to neladalkib. Ba/F3 cells expressing EML4–ALK variant 1 and different mutants were exposed to neladalkib for 72 h. Cell viability was evaluated by the CCK-8 assay, with absorbance measured at 450 nm. e Immunoblotting evaluation of the suppression of phosphorylated ALK expression in each resistant mutant by neladalkib. Ba/F3 cells expressing EML4–ALK variant 1 and different resistance mutations were treated with neladalkib for 3 h. Next, immunoblotting was used to detect the indicated protein in cell lysates. f–i Sensitivity of each resistance mutant to alectinib ( f ), crizotinib ( g ), lorlatinib ( h ), and brigatinib ( i ). Ba/F3 cells expressing EML4–ALK variant 1 and different resistance mutations were exposed to neladalkib for 72 h. Cell viability was evaluated by the CCK-8 assay, with absorbance measured at 450 nm. CCK-8 Cell Counting Kit-8, EML4 echinoderm microtubule-associated protein-like 4, ALK anaplastic lymphoma kinase, TKIs tyrosine kinase inhibitors

Article Snippet: Subsequently, the membranes were incubated overnight at 4℃ with gentle agitation in primary antibody dilution buffer (phosphorylated ALK [Y1604; Cell Signaling Technologies, Danvers, MA, USA; #3341, 1:1000], ALK [Cell Signaling Technologies; #3791, 1:2000], and α-tubulin [FUJIFILM Wako; #013-25033, 1:4000]).

Techniques: Mutagenesis, Variant Assay, Expressing, CCK-8 Assay, Western Blot, Cell Counting

Journal: Cell Death & Disease

Article Title: Targeting ERBB3 and AKT to overcome adaptive resistance in EML4-ALK-driven non-small cell lung cancer

doi: 10.1038/s41419-024-07272-7

Figure Lengend Snippet: Antibodies used for western blotting (WB) and dilutions.

Article Snippet: Anti-ALK (31F12) mouse mAb , CST , 3921 , 1:1000.

Techniques: Western Blot