Prediction of ALK mutations mediating ALK-TKIs resistance and drug re-purposing to overcome the resistance

Abstract

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.

Drug resistance mechanisms in Japanese anaplastic lymphoma kinase‐positive non–small cell lung cancer and the clinical responses based on the resistant mechanisms

“…MET gene amplification, which is well‐known as EGFR‐TKI resistance, a cause of bypass pathway activation‐mediated resistance to alectinib or ceritinib, was identified in 1 specimen. L1196M + G1202R, a compound mutation, was also a resistance mechanism to lorlatinib in patients with L1196M who previously experienced relapse while on crizotinib treatment …”

“…Second, secondary mutation may be observed more frequently in patients with crizotinib resistance in our cohort compared to previous reports, which may be due to the relatively higher plasma concentrations of crizotinib among Japanese ALK (+) patients than among Caucasian patients, as indicated by Fujiwara et al Third, additional mutation in the ALK kinase domain, which mediates acquired resistance to ALK‐TKI, usually emerges during crizotinib, ceritinib and alectinib failure, whereas a compound mutation in the kinase domain was the only resistance mechanism identified in lorlatinib failure. The compound mutation leading to lorlatinib failure has been reported by Shaw AT et al, Yoda et al and Okada et al in a preclinical model and in some clinical cases . Although Okada and his colleagues have shown that the use of early‐generation ALK‐TKI and several BCR‐ABL inhibitors might treat double mutation after lorlatinib failure, a strategy that can treat this type of resistance has not yet been established.…”

“…L1196M + G1202R, a compound mutation, was also a resistance mechanism to lorlatinib in patients with L1196M who previously experienced relapse while on crizotinib treatment. 25…”

Overcoming resistance by ALK compound mutation (I1171S + G1269A) after sequential treatment of multiple ALK inhibitors in non‐small cell lung cancer

“…Therefore, the search for drugs that can overcome these compound mutations is crucial and urgent. Several reports have shown that some of the lorlatinib‐resistant compound mutations became resensitized to first‐ or second‐generation ALK inhibitors (e.g., C1156Y + L1198F became resensitized to crizotinib, and I1171N + L1256F became resensitized to alectinib) …”

“…However, in several reports, diverse compound ALK mutations were identified in lorlatinib-resistant cells or patient samples after these patients had received the sequential ALK-TKI treatments. [7][8][9] The previously reported compound mutations found in patients are F1174V + G1202R, 10 C1156Y + L1198F, 7 E1210K + S1206C, E1210K + D1203N, 4 C1156Y + L1198F, I1171N + L1198F, L1171N + D1203N, L1196M + G1202R, G1202R + G1269A, D1203N + E1210K, G1202R + L1204V + G1269A, D1203N + E1210K + G1269A, 8 R1192P + G1202R, 11 G1202R + G1269A, 12 F1174L + G1202R, T1151M + G1202R, 13 L1196M + G1202R, I1171N + D1203N. 14 Therefore, the search for drugs that can overcome these compound mutations is crucial and urgent.…”