1. Resensitization to Crizotinib by the Lorlatinib ALK Resistance Mutation L1198F R1. 임빈 / prof. 김시영 2016.05.11

3. Introduction Small-molecule tyrosine kinase inhibitors -standard therapies for several types of cancer( chronic myeloid leukemia, epidermal growth factor receptor (EGFR)–mutated non–small-cell lung cancer (NSCLC), and ALK- rearranged NSCLC ) crizotinib -In NSCLC, ALK rearrangement, the first ALK inhibitor tested in the clinic -Resistance to crizotinib typically develops within the first year or two -Resistance : different mechanisms, including secondary mutations within the ALK tyrosine kinase domain and activation of alternative signaling pathways Lorlatinib -ATP-competitive small molecule inhibitor of ALK and the related tyrosine kinase ROS1 -3 rd generation -selectivity : the targeting of a specific residue in the ALK tyrosine kinase domain — leucine at position 1198 (L1198)

4. Cases report -A 52-year-old woman with metastatic ALK-rearranged NSCLC CT:new abdominal lymphadenopathy. lymph biopsy: crizotinib- resistant ALK C1156Y mutation. 41% reduction in tumor burden liver metastases worsening molecular testing revealed two ALK resistance mutations C1156Y, and L1198F

5. Method Patient -Informed consent to participate in the clinical trial. -Biopsies, molecular testing :in accordance with protocols approved by the institutional review board at Massachusetts General Hospital Genetic Studies -Screening for ALK rearrangement and amplification of the MET proto-oncogene (MET) : fluorescence in situ hybridization (FISH) -ALK resistance mutations: use of a targeted next-generation sequencing (NGS) assay and Sanger sequencing of complementary DNA Ba/F3 Cell-Line Studies -Ba/F3 cells: engineered to express echinoderm microtubule-associated protein-like 4 (EML4)– ALK harboring different ALK resistance mutations Biochemical and Structural Studies -Ki inhibition constants, kinetic characteristics, and co-crystal structures -co-crystal structures: the Worldwide Protein Data Bank

6. Result Genetic Analysis of Resistant Tumor Specimens ALK rearranged(30%) ALK, MET : no amplification -Targeted NGS-based PCR: two mutations in the ALK kinase domain: C1156Y, and L1198F C1156Y: known crizotinib-resistance mutation L1198F: gain-of-function mutation in anaplastic thyroid cancer and as a brigatinib-resistance mutation in combination with F1174V in a nucleophosmin-ALK–rearranged cell line -Similar frequencies :29% and 35%, respectively in the woman’s tumor -Same allele of the ALK fusion gene

7. Result Whole-exome sequencing -the double mutation (ALK C1156Y–L1198F) in the lorlatinib resistant sample -the single ALK C1156Y mutation in the crizotinib-resistant sample Clonal analysis

8. Cellular and Biochemical Characterization of Drug-Resistant ALK Mutants single ALK L1198F, C1156Y–L1198F => sensitive to crizotinib ALK single ALK L1198F, C1156Y–L1198F =>Greater binding affinity (crizotinib )

9. Result the enzymatic activity (Kcat/KM,substrate) C1156Y mutant 5.6 times as high as that of wild-type C1156Y–L1198F double mutant was mildly higher than that of the wild-type (1.7 times as high) ALK L1198F mutant was actually 2.5 times as low => alterations in drug binding, more so than changes in kinase activity, are probably the primary mechanism underlying resistance of C1156Y–L1198F to lorlatinib and sensitivity to crizotinib

10. Structural Studies of Drug-Resistant ALK Mutants cysteine at position 1156 (C1156) is located far (13 Å) from the inhibitor binding site and is not predicted to directly affect drug binding L1198 resides near the ATP binding site, and substitution of leucine with the larger phenylalanine =>steric clash with the nitrile of lorlatinib => not crizotinib (move slinghtly colser,more favorable by 0.8 kcal relative to wild-type ALK)

11. Conclusion Conclusion In cancer cells with the double mutation, the enhanced binding due to L1198F probably offsets the increased kinase activity due to C1156Y, leading to crizotinib sensitization.