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: Zotizalkib (TPX-0131) and gilteritinib (ASP2215) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

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: Calculation of infection efficiency a Cell growth after puromycin addition to calculate infection efficacy (zotizalkib, gilteritinib). The cell concentration at 0 h was 2 × 10 5 cells/mL. Living cells were counted at 42, 66, and 93 h after puromycin addition, and regression lines were drawn. The confidence of determination was 0.976 for zotizalkib (G1202R) and 0.997 for gilteritinib (I1171N), and the predicted initial infected cell concentration was 0.688 × 10 5 cells/mL for zotizalkib (G1202R) and 0.505 × 10 5 cells/mL for gilteritinib (I1171N). Consequently, the infection efficiency was calculated as 3.44% for zotizalkib (G1202R) and 2.53% for gilteritinib (I1171N). b Cell growth observation after puromycin addition to calculate infection efficacy (neladalkib). The cell concentration at 0 h was 2 × 10 5 cells/mL. Living cells were counted at 54, 68, and 80 h after puromycin addition, and regression lines were drawn. The confidence of determination was 0.937 for G1202R and 0.957 for I1171N, and the predicted initial infected cell concentration was 4.28 × 10 5 cells/mL for G1202R and 2.16 × 10 5 cells/mL for I1171N. Consequently, the infection efficiency was calculated as 21.4% for G1202R and 10.8% for I1171N.

Article Snippet: Zotizalkib (TPX-0131) and gilteritinib (ASP2215) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Infection, Concentration Assay

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: Zotizalkib (TPX-0131) and gilteritinib (ASP2215) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

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: Zotizalkib (TPX-0131) and gilteritinib (ASP2215) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

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: Structural mapping of resistance mutations on the ALK kinase domain a Structure of the ALK kinase domain highlighting key activation-related regions. b Docking pose of zotizalkib in the ALK kinase domain. c Spatial configuration of the phenylalanine core within the ALK kinase domain. d Docking pose of gilteritinib in the ALK kinase domain. e Docking pose of neladalkib in the ALK kinase domain. ALK anaplastic lymphoma kinase

Article Snippet: Zotizalkib (TPX-0131) and gilteritinib (ASP2215) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Activation Assay

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: Possible therapeutic strategies after alectinib failure. a Neladalkib is available for G1202R-harboring ALK-positive NSCLC, and no mutations were predicted to confer resistance to neladalkib. Zotizalkib is also available for G1202R-harboring ALK-positive NSCLC; however, additional resistance mutations such as F1174C/L/I/V may emerge. Each slice size in the compound mutation pie chart reflects the proportion of mutant clones obtained. b Gilteritinib is a treatment option for patients with I1171N-positive ALK-rearranged NSCLC. Most relapses may occur due to an additional E1210K or D1203N mutation. In such cases, neladalkib or repotrectinib may be effective. On the other hand, treatment of I1171N-mutant cells with neladalkib or ensartinib may lead to relapse due to the emergence of additional mutations such as L1256F, E1129V, or C1156Y; however, these compound mutants are considered to remain sensitive to gilteritinib. Sequential administration of gilteritinib-neladalkib or gilteritinib-ensartinib is expected to be beneficial for I1171N-positive relapses. Each slice size in the compound mutation pie chart reflects the proportion of mutant clones obtained. ALK anaplastic lymphoma kinase, NSCLC non-small cell lung cancer

Article Snippet: Zotizalkib (TPX-0131) and gilteritinib (ASP2215) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Mutagenesis, Clone Assay