The Ba/F3-EML4-ALK-L1256F cells grew without IL-3, suggesting that ALK-L1256F possessed oncogenicity. oligomerization of domains such as the coiled-coil domain of fusion partner. As a result, ALK downstream pathways, including the PI3K-AKT-mTOR, RAS-MAPK-ERK, or JAK-STAT pathways, are constitutively activated [3,4]. The ALK-tyrosine kinase inhibitor (TKI) crizotinib was first applied for the treatment of and in patients [10]. However, the G1202R mutation is resistant to first- and second-generation ALK inhibitors (crizotinib, alectinib, and ceritinib). The other second-generation ALK-TKI brigatinib was shown to be active against the G1202R mutant and [9]. Currently, although multiple ALK-compound mutants have been identified from lorlatinib sequential therapy resistant patients [12,13], the overcoming drugs against most of these mutants have not yet been clarified. To identify the lorlatinib or ceritinib resistance mechanisms in the ALK-G1202R or I1171N mutation-positive cancers, we performed N-ethyl-N-nitrosourea (ENU) mutagenesis screening and established a lorlatinib-resistant tumor using the EML4-ALK-G1202R mutation harboring mouse model. Molecular dynamic (MD) free energy simulation by the use of MP-CAFEE [14] successfully showed a clear linear correlation between experimental IC50 values of each ALK-TKI obtained using Ba/F3 cells expressing single- or QC6352 compound-mutated EML4-ALK QC6352 and the binding affinities of the ALK-TKI to the corresponding mutants. In addition, fragment molecular orbital (FMO) method [15] precisely quantified a marginal difference in the ALK-drug (alectinib) interaction among ALK mutants (I1171N, I1171N?+?L1256F, and L1256F), which could not be detected by the conventional MD simulation. Furthermore, we newly found and confirmed that L1256F single mutation confers marked resistance to lorlatinib but is extremely sensitive to alectinib. For a lorlatinib-resistant G1202R?+?L1196M double mutation, which is highly resistant to all ALK-TKIs, we found potential agents to suppress the resistant double mutation using high throughput drug screening. Our study models the possible lorlatinib-resistant compound mutations and shows potential therapeutic strategies to suppress this resistance. 2.?Materials and methods 2.1. Cell lines and reagents Human embryonic kidney cells, 293FT cells (Invitrogen), were cultured with Dulbecco’s Modified Eagle Medium high glucose (DMEM) supplemented with 10% fetal bovine serum and kanamycin (Meiji Seika Pharma, 250?mg/ml). And murine bone marrow derived pro-B cells, Ba/F3 cells, were cultured in DMEM low glucose supplemented with 10% fetal bovine serum, kanamycin and 0.5?ng/ml of interleukin-3 (IL-3). The cells were infected with retrovirus replicated in 293FT cells by transforming them with paging plasmids (pLenti), which contained rearranged cDNA regions encoding EML4-ALK variant 1 and either wild-type or different QC6352 resistance mutations (lorlatinib, ceritinib or alectinib). The pENTR/D-TOPO vector (Thermo Fisher Scientific) was used to clone the different cDNA regions by utilizing LR clonase II reactions; cells were selected with blastcidin (7?g/ml) for 1?week. After the selected cells grew, they were cultured in DMEM without IL-3. Alectinib- or ceritinib-resistant EML4-ALK (variants 3)-G1202R mutation-expressing patient-derived cell line JFCR-041-2 cells were cultured in StemPro hESC medium (Thermo Fisher Scientific) supplemented with 1 Antibiotic-Antimycotic Mixed Stock Solution (Nacalai tesque) and Y27632 (10?M). Alectinib-resistant EML4-ALK (variants 3)-I1171N mutation-expressing patient-derived cell line JFCR-043-2 cells were QC6352 cultured in media in which RPMI1640 (Thermo Fisher Scientific) and Ham’s F-12 (Nacalai tesque) were mixed in equal proportions, supplemented with 10% FBS and 1 Antibiotic-Antimycotic. Crizotinib (PF-02341066), lorlatinib (PF-06463922) or brigatinib (AP26113) were obtained from Shanghai Biochempartner. Alectinib (CH5424802) and ceritinib (LDK-378) was purchased from ActiveBiochem. 17-AAG was purchased from LC Laboratories. AG-957 was purchased from the Cayman Chemical Company. Adaphostin was purchased from SIGMA. Brigatinib was dissolved in ethanol for cell culture experiments. Other compounds were dissolved in dimethyl sulfoxide (DMSO) for cell culture. 2.2. Antibodies and.In addition, we identified several G1202R double mutations: G1202R?+?L1198F, +?L1196M, + F1174C, or +?F1174L. mutations and an L1256F single mutation as well as the potential therapeutic strategies for these ALK mutations. Our original computational simulation to calculate the binding affinity may be applicable for predicting resistant mutations and for overcoming drug resistance computational simulation to predict resistance conferred by kinase mutations and effective candidate drugs. Alt-text: Unlabelled Box 1.?Introduction In 2007, Soda and his colleagues found an (fusion gene from non-small-cell lung cancers (NSCLCs) [1]. the oligomerization of domains such as the coiled-coil domain of fusion partner. As a result, ALK downstream pathways, including the PI3K-AKT-mTOR, RAS-MAPK-ERK, or JAK-STAT pathways, are constitutively activated [3,4]. The ALK-tyrosine kinase inhibitor (TKI) crizotinib was first applied for the treatment of and in patients [10]. However, the G1202R mutation is resistant to first- and second-generation ALK inhibitors (crizotinib, alectinib, and ceritinib). The other second-generation ALK-TKI brigatinib was shown to be active against the G1202R mutant and [9]. Currently, although multiple ALK-compound mutants have been identified from lorlatinib sequential therapy resistant patients [12,13], the overcoming drugs against most of these mutants have not yet been clarified. To identify the lorlatinib or ceritinib resistance mechanisms in the ALK-G1202R or I1171N mutation-positive cancers, we performed N-ethyl-N-nitrosourea (ENU) mutagenesis screening and established a lorlatinib-resistant tumor using the EML4-ALK-G1202R mutation harboring mouse model. Molecular dynamic (MD) free energy simulation by the use of MP-CAFEE [14] successfully showed a clear linear correlation between experimental IC50 values of each ALK-TKI obtained using Ba/F3 cells expressing single- or compound-mutated EML4-ALK and the binding affinities of the ALK-TKI to the corresponding mutants. In addition, fragment molecular orbital (FMO) method [15] precisely quantified a marginal difference in the ALK-drug (alectinib) interaction among ALK mutants (I1171N, I1171N?+?L1256F, and L1256F), which could not be detected by the conventional MD simulation. Furthermore, we newly found and confirmed that L1256F single mutation confers marked resistance to lorlatinib but is extremely sensitive to alectinib. For a lorlatinib-resistant G1202R?+?L1196M double mutation, which is highly resistant to all ALK-TKIs, we found potential agents to suppress the resistant double mutation using high throughput drug screening. Our study models the possible lorlatinib-resistant compound mutations and shows potential therapeutic strategies to suppress this resistance. 2.?Materials and methods 2.1. Cell lines and reagents Human embryonic kidney cells, 293FT cells (Invitrogen), were cultured with Dulbecco’s Modified Eagle Medium high glucose (DMEM) supplemented with 10% fetal bovine serum and kanamycin (Meiji Seika Pharma, 250?mg/ml). And murine bone marrow derived pro-B cells, Ba/F3 cells, were cultured in DMEM low glucose supplemented with 10% fetal bovine serum, kanamycin and 0.5?ng/ml of interleukin-3 (IL-3). The cells were infected with retrovirus replicated QC6352 in 293FT cells by transforming them with paging plasmids (pLenti), which contained rearranged cDNA regions encoding EML4-ALK variant 1 and either wild-type or different resistance mutations (lorlatinib, ceritinib or alectinib). The pENTR/D-TOPO vector (Thermo Fisher Scientific) was used to clone the different cDNA regions by utilizing LR clonase II reactions; cells were selected with blastcidin (7?g/ml) for 1?week. After the selected cells grew, they were cultured in DMEM without IL-3. Cav3.1 Alectinib- or ceritinib-resistant EML4-ALK (variants 3)-G1202R mutation-expressing patient-derived cell line JFCR-041-2 cells were cultured in StemPro hESC medium (Thermo Fisher Scientific) supplemented with 1 Antibiotic-Antimycotic Mixed Stock Solution (Nacalai tesque) and Y27632 (10?M). Alectinib-resistant EML4-ALK (variants 3)-I1171N mutation-expressing patient-derived cell line JFCR-043-2 cells were cultured in media in which RPMI1640 (Thermo Fisher Scientific) and Ham’s F-12 (Nacalai tesque) were mixed in equal proportions, supplemented with 10% FBS and 1 Antibiotic-Antimycotic. Crizotinib (PF-02341066), lorlatinib (PF-06463922) or brigatinib (AP26113) were obtained from Shanghai Biochempartner. Alectinib (CH5424802) and ceritinib (LDK-378) was purchased from ActiveBiochem. 17-AAG was purchased from LC Laboratories. AG-957 was purchased from the Cayman Chemical Company. Adaphostin was purchased from SIGMA. Brigatinib was dissolved in ethanol for cell culture experiments. Other compounds were dissolved in dimethyl sulfoxide (DMSO) for.