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Success rate for re-biopsy in this study was approximately 80%. Our study sheds light on the re-biopsy status after disease progression in patients with advanced or metastatic NSCLC. This information is important to improve the selection of patients who may benefit from third-generation TKIs.

Background Alectinib has shown a greater efficacy to ALK -rearranged non-small-cell lung cancers in first-line setting; however, most patients relapse due to acquired resistance, such as secondary mutations in ALK including I1171N and G1202R. Although ceritinib or lorlatinib was shown to be effective to these resistant mutants, further resistance often emerges due to ALK-compound mutations in relapse patients following the use of ceritinib or lorlatinib. However, the drug for overcoming resistance has not been established yet. Methods We established lorlatinib-resistant cells harboring ALK-I1171N or -G1202R compound mutations by performing ENU mutagenesis screening or using an in vivo mouse model. We performed drug screening to overcome the lorlatinib-resistant ALK-compound mutations. To evaluate these resistances in silico , we developed a modified computational molecular dynamic simulation (MP-CAFEE). Findings We identified 14 lorlatinib-resistant ALK-compound mutants, including several mutants that were recently discovered in lorlatinib-resistant patients. Some of these compound mutants were found to be sensitive to early generation ALK-TKIs and several BCR-ABL inhibitors. Using our original computational simulation, we succeeded in demonstrating a clear linear correlation between binding free energy and in vitro experimental IC 50 value of several ALK-TKIs to single- or compound-mutated EML4-ALK expressing Ba/F3 cells and in recapitulating the tendency of the binding affinity reduction by double mutations found in this study. Computational simulation revealed that ALK-L1256F single mutant conferred resistance to lorlatinib but increased the sensitivity to alectinib. Interpretation We discovered lorlatinib-resistant multiple ALK-compound 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 in silico. Fund This work was mainly supported by MEXT/JSPS KAKENHI Grants and AMED Grants.

Proper attachment of microtubules to kinetochores is essential for accurate chromosome segregation. Here, we report a novel protein involved in kinetochore–microtubule attachment, chromosome alignment‐maintaining phosphoprotein (CAMP) (C13orf8, ZNF828). CAMP is a zinc‐finger protein containing three characteristic repeat motifs termed the WK, SPE, and FPE motifs. CAMP localizes to chromosomes and the spindle including kinetochores, and undergoes CDK1‐dependent phosphorylation at multiple sites during mitosis. CAMP‐depleted cells showed severe chromosome misalignment, which was associated with the poor resistance of K‐fibres to the tension exerted upon establishment of sister kinetochore bi‐orientation. We found that the FPE region, which is responsible for spindle and kinetochore localization, is essential for proper chromosome alignment. The C‐terminal region containing the zinc‐finger domains negatively regulates chromosome alignment, and phosphorylation in the FPE region counteracts this regulation. Kinetochore localization of CENP‐E and CENP‐F was affected by CAMP depletion, and by expressing CAMP mutants that cannot functionally rescue CAMP depletion, placing CENP‐E and CENP‐F as downstream effectors of CAMP. These data suggest that CAMP is required for maintaining kinetochore–microtubule attachment during bi‐orientation.

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