New pan-ALK inhibitor-resistant EML4::ALK mutations detected by liquid biopsy in lung cancer patients

Abstract

ALK and ROS1 fusions are effectively targeted by tyrosine kinase inhibitors (TKIs), however patients inevitably relapse after an initial response, often due to kinase domain mutations. We investigated circulating DNA from TKI-relapsed NSCLC patients by deep-sequencing. New EML4::ALK substitutions, L1198R, C1237Y and L1196P, were identified in the plasma of NSCLC ALK patients and characterized in a Ba/F3 cell model. Variants C1237Y and L1196P demonstrated pan-inhibitor resistance across 5 clinical and 2 investigational TKIs.

Fig. 1. Patients’ clinical data.

a Swimmer plot showing patients’ treatment and response, sorted by time on treatment. Pre-TKI therapy is not shown. A red diamond indicates the time of sampling. Bar colors represent TKIs, as indicated in the legend. TBP treatment beyond progression, PR partial response, SD stable disease, PD progressive disease, CHM chemotherapy, † death. b Number of wild-type (WT) and mutated (Mut) samples from subjects progressing on each inhibitor, excluding the one TBP sample. c Pie charts indicate the frequency of WT and Mut samples after crizotinib vs second-generation drugs (top) and after 1 vs > 1 TKI (bottom). Progression-free survival of WT vs Mut cases, considering the whole cohort (d) or ALK+ patients only (e).

Fig. 2. Effect of mutations on drug sensitivity.

a Diagram representing the EML4::ALK fusion, with a zoom on the kinase domain (KD), in which structural motifs are indicated. Mutations identified in this study are shown. b–i Cell viability and EML4::ALK phosphorylation of Ba/F3 cells expressing wild-type (WT, black lines) or mutant (colored lines) EML4::ALK, treated with the indicated TKIs. b, c L1198R from patient 2; d, e E1154K from patient 4; f, g C1237Y from patient 6; h, i L1196P from patient 14. (j–o) L1196P and C1237Y variants were tested with lorlatinib (j, k), zotizalkib (l, m) and repotrectinib (n, o). All data points represent mean ± s.e.m. Phospho-ALK and total ALK blots were run on the same membrane, after stripping.

Fig. 3. Molecular modelling of ALK mutants.

a Rotated views of ALK KD in complex with ADP (PDB: 3LCT) showing the position of the residues found mutated in this study (side chains shown as red sticks). The backbone is represented in green ribbon, except: αC-helix in cyan; αE-helix in lilac. ADP is in orange. b Superposition between ALK kinase domain (KD) in complex with crizotinib (PDB: 2XP2) in green and the model of R1198 after a 5 ns MD run. c RMSD values of the protein-ligand complexes for ALK kinase domain (KD) in complex with ADP (PDB: 3LCT; black line) and the corresponding C1237Y mutant (red). d Conformation of the ADP-bound C1237Y mutant highlighting the H bond network across Y1237-H1247-D1270 residues. e Close view of lorlatinib (shown as filled space) binding within WT (L1196; left) and mutant (P1196; right) ALK KD. The mutation disrupts a hydrophobic interaction of lorlatinib with the hinge region.