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Analysis of lorlatinib analogs reveals a roadmap for targeting diverse compound resistance mutations in ALK-positive lung cancer

Abstract

Lorlatinib is currently the most advanced, potent and selective anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor for the treatment of ALK-positive non-small cell lung cancer in the clinic; however, diverse compound ALK mutations driving therapy resistance emerge. Here, we determine the spectrum of lorlatinib-resistant compound ALK mutations in patients, following treatment with lorlatinib, the majority of which involve ALK G1202R or I1171N/S/T. We further identify structurally diverse lorlatinib analogs that harbor differential selective profiles against G1202R versus I1171N/S/T compound ALK mutations. Structural analysis revealed increased potency against compound mutations through improved inhibition of either G1202R or I1171N/S/T mutant kinases. Overall, we propose a classification of heterogenous ALK compound mutations enabling the development of distinct therapeutic strategies for precision targeting following sequential tyrosine kinase inhibitors.

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Fig. 1: Spectrum of compound ALK mutations identified in lorlatinib-resistant biopsies.
Fig. 2: Drug screening of Ba/F3 ALK mutation models identifies LAs with increased potency against compound ALK mutations.
Fig. 3: Selectivity of LA7 and LA9 against I1171N and G1202R single and compound mutants, respectively.
Fig. 4: Differential selectivity of LA7 and LA9 in patient-derived ALK-positive NSCLC cell lines.
Fig. 5: LA7 and LA9 inhibit tumor growth in xenograft tumors bearing ALK compound mutations.
Fig. 6: Cellular IC50 changes with single and compound ALK mutations.
Fig. 7: Structural basis for selectivity of LA7 and LA9 against ALK compound mutations.

Data availability

The crystal structural data and methods have been deposited to the Protein Data Bank (PDB) and have been assigned ID codes: lorlatinib, PDB 4CLI;14 LA7, PDB 7R7R (1.93 Å); and LA9, PDB 7R7K (1.83 Å). Source data have been provided as Source Data files. All other data supporting the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank patients and their families, as well as members of the Hata laboratory and MGH Thoracic Oncology Group for helpful discussion and support. This study was supported by a JSPS Overseas Research Fellowships (to A.S.), a National Cancer Institute Career Development Award (K12CA087723–16 to I.D.J.), NIH/NCI R01CA164273 (to J.J.L.), a Lung Cancer Research Foundation grant (to S.Y.), by Be a Piece of the Solution and by Targeting a Cure for Lung Cancer Research Fund at MGH.

Author information

Authors and Affiliations

Authors

Contributions

A.S.I., T.W.J., T.R.J., M.A.M., M.A.W., L.N.P., K.D., A.A., J.L.B. and S.Y. performed the experiments. I.D.J., M.M.K., L.A.K., A.D., J.L.P., J.F.G., J.J.L. and A.N.H. contributed human samples and/or data. A.S.I., T.W.J., I.D.J., M.M.K., T.R.J., P.W., S.L.W., M.A.M., L.A.K., B.Y.Y., J.J.L., S.Y. and A.N.H. performed data analysis and interpretation. A.S.I., T.W.J., I.D.J., T.R.J., M.A.M., L.K., B.Y.Y., J.J.L., S.Y. and A.N.H. wrote the manuscript. J.J.L., S.Y. and A.N.H. jointly supervised the work.

Corresponding authors

Correspondence to Jessica J. Lin, Satoshi Yoda or Aaron N. Hata.

Ethics declarations

Competing interests

T.W.J., T.R.J., P.W., S.L.W. and M.A.M. are employees of Pfizer. I.D.J. has received honoraria from Foundation Medicine, Creative Education Concepts and American Lung Association, consulting fees from Guidepost, AstraZeneca, Boehringer Ingelheim, BostonGene, Catalyst, Novocure, Pfizer, Syros and Xcovery, research support from Array, Genentech, Novartis, Pfizer and Guardant Health and travel support from Array and Pfizer. L.A.K. is an employee and shareholder of Guardant Health. J.F.G. has served as a compensated consultant or received honoraria from Bristol-Myers Squibb, Genentech, Ariad/Takeda, Loxo, Pfizer, Incyte, Novartis, Merck, Agios, Amgen, Jounce, Karyopharm, GlydeBio, Regeneron, Oncorus, Helsinn, Jounce, Array and Clovis Oncology, has an immediate family member who is an employee with equity in Ironwood Pharmaceuticals, has received research funding from Novartis, Genentech/Roche and Ariad/Takeda and institutional research support from Tesaro, Moderna, Blueprint, BMS, Jounce, Array, Adaptimmune, Novartis, Genentech/Roche, Alexo and Merck. J.J.L. has served as a compensated consultant for Genentech, C4 Therapeutics, Blueprint Medicines, Nuvalent, Turning Point Therapeutics, Mirati Therapeutics, Bayer and Elevation Oncology; received honorarium and travel support from Pfizer; received institutional research funds from Hengrui Therapeutics, Turning Point Therapeutics, Neon Therapeutics, Relay Therapeutics, Bayer, Elevation Oncology, Roche/Genentech, Pfizer, Nuvalent, Linnaeus Therapeutics and Novartis; received continuing medical education funding from OncLive, MedStar Health and Northwell Health. S.Y. is an employee of Tango Therapeutics and has received a consulting fee from Pfizer Japan and Nuvalent. A.N.H. has received research support from Pfizer, Nuvalent, Roche/Genentech, Amgen, Blueprint Medicines, Eli Lilly, Scorpion Therapeutics, Bristol-Myers Squibb, BridgeBio and Relay Therapeutics; has served as a compensated consultant for Nuvalent, Tolremo Therapeutics, Engine Biosciences and TigaTx. The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Clinical outcomes on lorlatinib.

(A) Time to progression on lorlatinib according to presence (N = 13 patients) or absence (N = 8 patients) of a baseline ALK mutation. (B) Time to treatment discontinuation from lorlatinib according to the presence (N = 13 patients) or absence (N = 8 patients) of a baseline ALK mutation. Only those patients with available pre-lorlatinib tissue biopsy and known status of baseline ALK mutations are included in these analyses. Both time to progression and time to treatment discontinuation curves were estimated using the Kaplan-Meier method.

Extended Data Fig. 2 Sensitivity of compound ALK mutations to currently FDA-approved ALK tyrosine kinase inhibitors.

Cellular IC50 values (A) and dose response curves (B) of Ba/F3 cells expressing clinical ALK compound mutations to crizotinib, ceritinib, brigatinib, alectinib, and lorlatinib. Data correspond to the heatmap shown in Fig. 1E. Data are mean and SEM of three independent experiments.

Extended Data Fig. 3

Molecular structures of lorlatinib analogs (LAs) 1–20.

Extended Data Fig. 4 Drug screening with lorlatinib analogs using Ba/F3 ALK mutation models.

(A) Cellular IC50 values of each LA against single ALK mutations (top) and Ba/F3 parental and PC9 cells (bottom). This data corresponds to the heatmap in Fig. 2B (N = 1). When the calculated IC50 exceeded the highest concentration used in the experiment (10μM), the value was plotted as >10000. (B) Cellular IC50 values of 12 selected LAs against clinical ALK compound mutations. This data corresponds to the heatmap in Fig. 2C (N = 1). (C) Cellular IC50 values of 6 selected LAs against an expanded set of clinical and putative compound ALK mutations. This data corresponds to the heatmap in Fig. 2D (N = 1).

Extended Data Fig. 5 Drug dose response curves of lorlatinib analogs against Ba/F3 ALK mutation models.

Cell viability assays performed with Ba/F3 cells expressing EML4-ALK with the indicated compound mutations treated with 6 LAs or lorlatinib for 48 hours. The viabilities were measured with CellTiter-Glo assay. These data correspond to the heatmap in Fig. 2D and dot plots in Extended Data Fig. 4C. The experiments were performed once.

Extended Data Fig. 6 Distinct activity of LA7 and LA9 in Ba/F3 models and the patient-derived models.

A) Western blot analysis performed on compound mutation Ba/F3 models. Ba/F3 cells expressing nonmutant EML4-ALK, EML4-ALK G1202R + G1269A, G1202R + S1206F + G1269A or I1171N + L1198F were treated with DMSO, LA7, LA9, or lorlatinib for 6 hours, and total cell lysates were analyzed by western blotting. The images are representative of at least two repeats with similar results. (B) Comparison of LA7 and LA9 with currently approved ALK inhibitors. Cellular IC50 values of LA7, LA9 and currently approved ALK inhibitors including lorlatinib against clinical ALK compound mutations. IC50 values for approved ALK inhibitors are replotted from Extended Data Fig. 2A for comparison purposes. Data are mean of three independent experiments. (C) MGH953-4 (G1202R) cells were treated with the indicated drugs for 5 days and cell viability assessed with CellTiter-Glo assay. Data are mean and SEM of three independent experiments. (D) Cellular IC50 values of LA7 and LA9 compared to approved ALK inhibitors in MGH953-4, MGH953-7 (G1202R + L1196M), and MGH990-2 (I1171N + D1203N) cells. Data are mean of three independent experiments. (E) The ratio of cell viability with LA7 or LA9 in MGH953-7 (G1202R + L1196M) at 300 nM or MGH990-2 (I1171N + D1203N) at 100 nM, corresponding to Fig. 4D. Data are mean and SEM of three independent experiments.

Source data

Extended Data Fig. 7 In vivo activity of LA7 and LA9.

(A) Body weight of mice bearing MGH953-7 during the course of treatment (N = 6 mice per group). (B) Body weight of mice bearing NIH3T3 EML4-ALK I1171N + D1203N xenograft tumors during the course of treatment (N = 6 per group). (C) Change in tumor volume of NIH3T3 G1202R + L1196M xenograft tumors. Mice were treated with lorlatinib 6 mpk QD, LA7 20 mpk QD, or LA9 40 mpk QD (N = 6 mice per group). Tumors treated with LA9 were significantly smaller on day 8 compared to those with vehicle (p = 0.048, two-tailed t-test). (D) Body weight of mice bearing NIH3T3 EML4-ALK I1171N + D1203N xenograft tumors during the course of treatment (N = 6 mice per group). (E) Western blot analysis of NIH3T3 cells expressing ALK G1202R + L1196M and treated with the indicated drugs for 1 hour. (F) The ratio of tumor growth inhibition (TGI) with LA7 and LA9 in NIH3T3 EML4-ALK I1171N + D1203N or G1202R + L1196M xenograft tumors, corresponding to Fig. 5D and Extended Data Fig. 7C. Error bars indicate SEM for 6 mice per group. (G) Unbound systemic concentrations of lorlatinib, LA7 and LA9 were calculated based on the total drug concentrations in whole blood measured in vivo and the unbound fraction in blood (fu,b; calculated as fu,p / BPR). Data are mean and SEM of three mice.

Source data

Extended Data Fig. 8 Phospho-ALK immunohistochemistry of MGH953-7 PDX tumors and control specimens.

(A) MGH953-7 PDX tumors treated with lorlatinib, LA7 or LA9 were harvested after 3-day treatment and FFPE sections were stained with anti-phospho-ALK antibody. Scale bar in low magnification images: 300μm. Scale bar in high magnification images: 50μm. (B) Patient-derived xenografts (PDXs) of two EGFR-mutated NSCLC were used as negative controls and PDXs of two ALK-positive NSCLC were used as positive controls. FFPE sections were stained with anti-phospho-ALK antibody. Phospho-ALK is localized in the cytoplasm and exhibits a diffuse staining pattern in tumor cells. Scale bar in low magnification images: 300μm. Scale bar in high magnification images: 20μm.

Extended Data Fig. 9 Relative changes in IC50 values for lorlatinib, LA7 and LA9 against single and compound ALK mutations.

(A) Lorlatinib, LA7 and LA9 cellular IC50 values (upper left) and IC50 ratios (lower right) of single mutant vs nonmutant ALK (left panel) or compound mutant vs single mutant ALK (middle and right panels). IC50 values correspond to data shown in the Source Data. (B) Fold improvement of LA7 or LA9 compared to lorlatinib against single and compound mutations (calculated by dividing the lorlatinib IC50 by the LA IC50). (C) Cell viability assays performed with Ba/F3 cells expressing nonmutant EML4-ALK with 0.01–100 nM lorlatinib, LA7 or LA9 for 48 hours. The viabilities were measured with CellTiter-Glo assay. Data are mean and SEM of three independent experiments.

Extended Data Fig. 10 Structural basis for selectivity of lorlatinib analogs against ALK compound mutations and the L1198F mutation.

(A) Energy-minimized model of LA9 bound to G1202R/L1196M. Solvent front G1202R and D1203 residues are bolded for emphasis. Productive interactions shown by dotted lines are similar to G1202R single mutant (see Fig. 7B). (B) Co-crystal structure of LA7 bound to wild-type ALK (upper) and energy minimized models of LA7 bound to I1171N (middle) and I1171N/D1203N (bottom) superimposed with energy-minimized model of I1171N (ligand and protein colored pink) showing that the network of hydrogen bonds between thiazole ring hydroxyl groups and solvent front residues are largely preserved. (C) Energy minimized model of LA7 bound to G1202R/L1196M showing disruption of solvent front hydrogen bond network, similar to G1202R single mutant (see Fig. 7D). (D)-(G) The L1198F mutation was modeled onto co-crystal structures of lorlatinib (D), LA7 (E), LA9 (F) or LA4 (G) bound to WT ALK. The L1198F substitution results in steric clash with the selectivity nitrile of lorlatinib. LA7 and LA9 lack the selectivity nitrile and can easily accommodate the L1198F substitution, whereas the nitrile of LA4 is positioned toward L1198F but exhibits reduced steric clash compared to lorlatinib due to the more flexible ligand structure. (H) Relative fold potency decrease of LA4 compared with LA7 (calculated by dividing the cellular IC50 values of LA4 by the that of LA7) against Ba/F3 models harboring compound ALK mutants. IC50 values correspond to data shown in the Source Data. (I) Superimposition of LA7 onto the LA4/L1198F model shown in Panel G comparing the position of the corresponding thiazole methyl or nitrile groups near L1198F.

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Statistical Source Data.

Source Data Fig. 2

Numerical Source Data.

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Source Data Fig. 3

Unprocessed western blots.

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Source Data Extended Data Fig. 6

Statistical Source Data.

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Unprocessed western blots.

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Shiba-Ishii, A., Johnson, T.W., Dagogo-Jack, I. et al. Analysis of lorlatinib analogs reveals a roadmap for targeting diverse compound resistance mutations in ALK-positive lung cancer. Nat Cancer 3, 710–722 (2022). https://doi.org/10.1038/s43018-022-00399-6

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