Fig 2: Identification of inhibitors able to inhibit the lorlatinib-resistant ALK-G1202R + L1196M double mutant.(a) Relative cell viability of Ba/F3 parental cells, Ba/F3 poly clonal cells expressing EML4-ALK-WT, EML4-ALK-G1202R, or EML4-ALK-G1202R + L1196M for AG-9571000 nM. (b) The structure of AG-957. (c) The structure of adaphostin. (d and e) Ba/F3 parental cells, Ba/F3 poly clonal cells expressing EML4-ALK-WT, Ba/F3 poly clonal cells expressing EML4-ALK-G1202R, and Ba/F3 ALK-G1202R + L1196M clonal cells #50 were treated with the indicated concentrations of AG-957 (d) or adaphostin (e) for 72 h. Cell viability was analyzed using the CellTiter-Glo assay. (f) Calculated IC50 values of parental Ba/F3 cells, EML4-ALK-WT Ba/F3 cells, EML4-ALK-G1202R Ba/F3 cells, or EML4-ALK-G1202R + L1196M Ba/F3 clonal cells #50 are shown. These cells were treated with AG-957, or adaphostin for 72 h. Results are expressed as mean ± SD. (g) Average IC50 values (from three independent experiments) of each Ba/F3 cell to AG-957 or adaphostin are shown. (h) Suppression of phospho-ALK, AKT, or S6 in each Ba/F3 cell by AG-957 or adaphostin. Cells were exposed to increasing concentrations (AG-957: 300 nM and 1000 nM; adaphostin: 300 nM, 600 nM, and 1000 nM) of each inhibitor for 3 h. Cell lysates were immunoblotted to detect the indicated proteins.

Fig 3: Identification of lorlatinib-resistant G1202R + G1269A double mutation in a mouse xenograft model using patient-derived ALK-G1202R positive cells.(a) Graphic depicting the scheme to establish the lorlatinib-resistant tumor is presented. (b) The sequence of JFCR-041-2 LorR cells. (c) Suppression of phospho-ALK and its downstream signaling in JFCR-041-2 or JFCR-041-2 LorR cells by ALK-TKI treatment. Cells were exposed to 300 nM of each ALK-TKI for 3 h. Cell lysates were immunoblotted to detect the indicated proteins. (d and e) Suppression of phospho-ALK and its downstream signaling by lorlatinib (d) or brigatinib (e) in each Ba/F3 cells. Cells were exposed to increasing concentrations (10 nM, 100 nM, 300 nM, 600 nM and 1000 nM) of each inhibitor for 3 h. Cell lysates were immunoblotted to detect the indicated proteins.

Fig 4: Computational simulation of the binding affinity between ALK compound mutants and ALK-TKIs by MP-CAFEE and FMO calculations.(a and b) The calculate ΔG values of lorlatinib (a), or crizotinib (b) to each ALK double mutant are shown in the bar graph. (c, d) MD-relaxed structures of alectinib-bound ALK-WT (green), I1171N (cyan), I1171N + L1256F (magenta), and L1256F single (orange). I/N1171, L/F1256, and alectinib in an energetically stable conformation obtained from five sets of 50 ns simulations are depicted by sticks (green/cyan/magenta, carbon; blue, nitrogen; red, oxygen). Average ΔG values (from three sets of the free energy simulations) are indicated. (e–g) The position of L/F1256 and alectinib in the tertiary structure of (e) ALK-I1171N (cyan), (f) ALK-I1171N + L1256F (magenta), or (g) ALK-L1256F single (orange) mutant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig 5: Sequencing data from the next‐generation sequencing (NGS) and Sanger sequencing. (a) Next‐generation sequencing shows nucleotide 3512 t > g mutation and 3806 g > c mutation. (b) cDNA from the total RNA of the liver biopsy sample was used to amplify ALK by PCR, and Sanger sequencing of the ALK kinase domain showed nucleotide 3512 t > g mutation and 3806 g > c mutation as major peak.