Diverse Resistance Mechanisms to the Third-Generation ALK Inhibitor Lorlatinib in ALK-Rearranged Lung Cancer

Abstract

Purpose: Lorlatinib is a third-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor with proven efficacy in patients with ALK-rearranged lung cancer previously treated with first- and second-generation ALK inhibitors. Beside compound mutations in the ALK kinase domain, other resistance mechanisms driving lorlatinib resistance remain unknown. We aimed to characterize the mechanisms of resistance to lorlatinib occurring in patients with ALK-rearranged lung cancer and design new therapeutic strategies in this setting.

Experimental design: Resistance mechanisms were investigated in 5 patients resistant to lorlatinib. Longitudinal tumor biopsies were studied using high-throughput next-generation sequencing. Patient-derived models were developed to characterize the acquired resistance mechanisms, and Ba/F3 cell mutants were generated to study the effect of novel ALK compound mutations. Drug combinatory strategies were evaluated in vitro and in vivo to overcome lorlatinib resistance.

Results: Diverse biological mechanisms leading to lorlatinib resistance were identified. Epithelial-mesenchymal transition (EMT) mediated resistance in two patient-derived cell lines and was susceptible to dual SRC and ALK inhibition. We characterized three ALK kinase domain compound mutations occurring in patients, L1196M/D1203N, F1174L/G1202R, and C1156Y/G1269A, with differential susceptibility to ALK inhibition by lorlatinib. We identified a novel bypass mechanism of resistance caused by NF2 loss-of-function mutations, conferring sensitivity to treatment with mTOR inhibitors.

Conclusions: This study shows that mechanisms of resistance to lorlatinib are diverse and complex, requiring new therapeutic strategies to tailor treatment upon disease progression.

Figure 1

Summary of ALK-rearranged patient included in the MATCH-R study. NSCLC: non-small cell lung cancer, EMT: epithelial mesenchymal transition

Figure 2

SRC and ALK inhibition overcomes lorlatinib resistance mediated by EMT. A, Treatment course of patient MR57 (PR, partial response). B, MR57-S and MR57-R cells were treated with increasing concentrations of lorlatinib for 24hs. Cell lysates were immunoblotted to detect the selected proteins. C, Treatment course of patient MR210 (PD, progressive disease). D, Phenotype of MR210 epithelial and mesenchymal cells labelled with Cy3 Phalloidin and DAPI. E, MR57-R cells were treated with the indicated doses of lorlatinib and saracatinib alone or in combination, for 7 days. Cell viability was assessed with Cell Titer Glo. F, MR210 cells were treated with single agents lorlatinib, saracatinib, erdafitinib and debio-1347 or in combination for 7 days. Cell viability was assessed with Cell Titer Glo. G, MR57 lorlatinib sensitive (epithelial) and resistant (mesenchymal) cells were treated with the specified concentrations of lorlatinib and saracatinib for 24hs. Cell lysates were probed with antibodies against the indicated proteins. H, Phenotypes of MR57 epithelial and mesenchymal cells labelled with Alexa Fluor 488 Phalloidin and DAPI after treatment with lorlatinib and saracatinib for 30 days.

Figure 3

Resistance to lorlatinib mediated by ALK kinase domain compound mutations. A, Clinical course of patient MR144 and allelic frequencies of ALK resistant mutations (from RNA sequencing) with sequential treatments. B, Fish Plot illustrating the tumor clonal evolution obtained by WES analysis during treatment with ALK inhibitors. The ALK E1154K and G1202R subclones emerged independently upon resistance to crizotinib. After disease progression with brigatinib, the ALK G1202R clone predominated and the E1154K clone became undetectable. At lorlatinib resistance, a subclone emerged from the ALK G1202R clone acquiring an additional F1174L mutation. C, Clinical course of patient MR347. D, Cell survival assay of Ba/F3 models with the indicated ALK single and the F1174L/G1202R compound mutations treated with lorlatinib for 48hs. E, Cell survival assay of Ba/F3 models with the indicated ALK single and the L1196M/D1203N compound mutations treated with lorlatinib for 48hs. F, ALK and downstream kinases phosphorylation in Ba/F3 mutated cells treated with the indicated concentrations of lorlatinib for 3hs. G, Direct comparison of ALK phosphorylation in the same Ba/F3 models by immunoblotting of cell lysates after 3hs treatment with lorlatinib showing higher levels of ALK phosphorylation with the F1174L/G1202R compound mutation. H, Visual representation of aligned wild-type (green) and F1174L/G1202R mutated (brown) ALK structures in complex with lorlatinib.

Figure 4

NF2 loss of function mediates resistance to lorlatinib. A, Clinical course of patient MR135 and mutational profile of samples obtained on lorlatinib progression (PD, progressive Disease). B, Cell survival assay assessed with Cell Titer Glo of MR135 lorlatinib resistant cells from biopsy 1 (MR135-R1) treated for 7 days with the indicated concentrations of lorlatinib and vistusertib (AZD2014) alone or in combination. C, Immunoblot analysis from cell lysates of MR135-R1 treated for 24hs with the specified doses of lorlatinib, vistusertib (AZD2014) and ponatinib alone or in combination using indicated antibodies. D, Athymic nude mice bearing MR135-R2 PDX were administered lorlatinib or vistusertib 20 mg/kg orally. Tumor volumes, mean ±SD (n =8); (*** p < 0.001). E, Cell lysates from H3122 parental and H3122 cells with NF2 heterozygous deletions or homozygous deletions, generated by CRISPR-CAS9 gene editing, were immunoblotted to detect merlin expression. H3122 cells with bi-allelic NF2 knock-out lacked merlin expression. F, Cell survival assay of H3122 parental and H3122 NF2 knock-out (NF2 KO) cells treated with lorlatinib for 7 days. Cell survival was assessed by Cell Titer Glo. G, Cell proliferation assay of H3122 parental and H3122 NF2 KO cells untreated and treated with lorlatinib measured at baseline, day 2, day 5 and day 7. Cell viability was assessed with Cell Titer Glo. H, Caspase 3/7 activation (Caspase 3/7-Glo assay) relative to the number of live cells simultaneously assessed in the cell proliferation assay previously described. I, H3122 parental and NF2 KO cells were treated with the indicated doses of lorlatinib for 24hs. Cell lysates were immunoblotted to detect the selected proteins.