Gilteritinib overcomes lorlatinib resistance in ALK-rearranged cancer

Abstract

ALK gene rearrangement was observed in 3%–5% of non-small cell lung cancer patients, and multiple ALK-tyrosine kinase inhibitors (TKIs) have been sequentially used. Multiple ALK-TKI resistance mutations have been identified from the patients, and several compound mutations, such as I1171N + F1174I or I1171N + L1198H are resistant to all the approved ALK-TKIs. In this study, we found that gilteritinib has an inhibitory effect on ALK-TKI–resistant single mutants and I1171N compound mutants in vitro and in vivo. Surprisingly, EML4-ALK I1171N + F1174I compound mutant-expressing tumors were not completely shrunk but regrew within a short period of time after alectinib or lorlatinib treatment. However, the relapsed tumor was markedly shrunk after switching to the gilteritinib in vivo model. In addition, gilteritinib was effective against NTRK-rearranged cancers including entrectinib-resistant NTRK1 G667C-mutant and ROS1 fusion-positive cancer.

Deciphering the Mechanism of Gilteritinib Overcoming Lorlatinib Resistance to the Double Mutant I1171N/F1174I in Anaplastic Lymphoma Kinase

“…That is to say, in the wild-type system, lorlatinib and gilteritinib bound to the ALK kinase domain with a similar ability. This prediction was in consistent with the experimental CellTiter-Glo assays that the IC 50 value of lorlatinib (1.2 ± 0.38 nM) and gilteritinib (0.78 ± 0.27 nM) to ALK was nearly the same, suggesting the equal inhibitory capacity of ALK by lorlatinib and gilteritinib ( Mizuta et al, 2021 ). In the double mutant of gilteritinib-bound state, the predicted binding free energy was slightly higher by 2.07 kcal/mol than that of the counterpart wild-type system.…”

“…Significantly, when lorlatinib bound to the double mutant, the predicted binding free energy caused an increase of 7.67 kcal/mol in relation to the corresponding wild-type system. Consistently, the experimental IC 50 of lortatinib to the double mutant I1171N/F1174I was 338 ± 41 nM, which increased by ∼282-fold compared to the wild-type ALK ( Mizuta et al, 2021 ). Therefore, according to the energetical prediction results, gilteritinib could still bind to the double mutant ALK I1171N/F1174I, albeit with a relatively decreased binding affinity respective to the wild-type state, whereas the double mutations had a highly detrimental effect on the ability of ALK to bind lorlatinib.…”

“…In the double mutant of gilteritinib-bound state, the predicted binding free energy was slightly higher by 2.07 kcal/mol than that of the counterpart wild-type system. Indeed, the experimental IC 50 of gilteritinib to the double mutant I1171N/F1174I was 24 ± 4.4 nM, which increased by ∼30-fold compared to the wild-type ALK ( Mizuta et al, 2021 ). Significantly, when lorlatinib bound to the double mutant, the predicted binding free energy caused an increase of 7.67 kcal/mol in relation to the corresponding wild-type system.…”

“…Gilteritinib is an ATP-competitive tyrosine multi-kinase inhibitor that has been approved for treating relapsed or refractory Fms-like tyrosine kinase 3 (FLT3)-positive acute myeloid leukemia (AML) ( Pulte et al, 2021 ). Multiple experiments containing the ALK kinase activity, phosphoproteomic analysis of ALK-positive lung cancer cells, and kinase substrate-enrichment analysis unequivocally ascertained that gilteritinib directly inhibited the growth of ALK -rearranged human NSCLC cells and further overcame lorlatinib resistance to the double mutant ALK I1171N/F1174I ( Mizuta et al, 2021 ). These data indicated that in addition to treat relapsed or refractory FLT3-positive AML, gilteritinib could also function as drug repositioning, that is, treating for ALK-positive NSCLC including lorlatinib-resistant ALK I1171N/F1174I double mutant.…”

“…To overcome the double mutation I1171N/F1174I, a further inhibitor library screening identified a gilteritinib inhibitor that suppressed the viability of both wild-type and the double I1171N/F1174I mutant expressing Ba/F3 cells ( Mizuta et al, 2021 ) ( Figure 1C ). Currently, the unavailability of a crystal structure of gilteritinib to the ALK kinase domain caused the poor understanding the molecular mechanism of how gilteritinib overcomes lorlatinib resistance in the double mutant ALK I1171N/F1174I.…”