a/f3 G1del ell viability (% control) a/f3 G1R ell viability (% control) Figure S1 MGH9-1 (iopsy) MGH9-1 (ell Line) G1del LK G1del (c.33_35delggg) Reference MGH9-1 D 5 rizotinib I 5 = 5. nm eritinb I 5 =.1 nm Lorlatinib I 5 = 5. nm lectinib I 5 = 75. nm rigatinib I 5 = 5. nm 5 rizotinib I 5 = 51 nm eritinb I 5 = 17. nm Lorlatinib I 5 =. nm lectinib I 5 = 359 nm rigatinib I 5 = 7. nm 1 Dose (nm) 1 Dose (nm) Fig. S1: LK G1del is a novel resistance mutation in LK-rearranged lung cancer:. Pile-up of the LK G1del identified by next-generation sequencing in a lung biopsy specimen from a patient progressing on ceritinib.. onfirmation of an LK G1del mutation in a corresponding patient-derived cell line (MGH9-1) using Sanger sequencing of complementary DN. - D. ell viability of a/f3 cells harboring EML-LK G1del and G1R, respectively, treated with first-, second- and thirdgeneration LK inhibitors.
Residual plk level ctrl 3 3 3 3 3 3 3 3 ctrl 3 3 3 3 3 3 3 3 Figure S EML-LK WT a/f3 hr treatment G1del a/f3 hr treatment rizotinib eritinib Lorlatinib lectinib rizotinib eritinib Lorlatinib lectinib nm: nm: plk plk 1 1 plk 1 T/Y T/Y T/Y T/Y ps ps ps ps GPDH GPDH GPDH 5 Dose (nm) WT rizotinib G1del rizotinib WT eritinib G1del eritinib WT lectinib G1del lectinib WT Lorlatinib G1del Lorlatinib Fig. S: LK G1del in LK-rearranged lung cancer: -. a/f3 cells transformed by wildtype or G1del EML-LK, respectively, were treated with the indicated concentrations of crizotinib, ceritinib, alectinib and lorlatinib for hours. Lysates were probed with antibodies directed against the specified proteins.. Dose response western blot curves show LK phosphorylation levels in EML-LK WT and G1del a/f3 cells treated with indicated doses of crizotinib, ceritinib, alectinib and lorlatinib for hours. Error bars indicate SEMs of three independent experiments.
Figure S3 MGH935 17 MGH917 1 MGH7 15 MGH9 1 LK G1R LK WT LK L119M LK G1R MGH3 13 MGH9 1 MGH97 11 LK WT LK WT LK WT MGH7 MGH93 9 MGH9 MGH913 7 MGH9 MGH5 5 MGH91 MGH99 3 MGH9 MGH919 1 LK WT a -> LK V11L b LK G1R LK G1R LK I1171S LK WT a -> LK WT b LK I1171T LK WT LK WT LK WT a -> LK WT b LK G1R Series1 rizotinib Series Interval Therapy Series3 lectinib iopsy 3 5 Duration of Treatment (Months) Fig. S3: Swimmer s plots depicting LK inhibitor treatment courses and biopsy time-points for patients with alectinib-resistant biopsies. Therapies received prior to crizotinib are not depicted. Intervening therapies were received by four patients and included ceritinib (MGH97, MGH9, and MGH9) and pemetrexed/crizotinib (MGH9). LK WT refers to absence of an LK resistance mutation. mong patients with two separate biopsies, (a) indicates findings in a post-crizotinib biopsy and (b) indicates findings in a post-alectinib biopsy.
Figure S Fold hange GI5 1 T3 EW51 fatinib IR P113 Z Z9 ZD53 ZD115 ZD ay19 EZ35 GJ-39 I53 MS- ortezomib YL719 GP-7 rizotinib T991 yclopam Dacomiti Dasatinib Dovitinib Elesclomol Embelin Flavopiridol GD9 GD91 Gefitnib Geldana GSK99 GSK539 GW57 I711 Imatinib KIN1-113 KIN1-135 KU55933 Lapatinib LW Lestaurtinib LFM13 Linsitinib MGD5 Midostau MK MLN37 MRK3 Olaparib PD33991 PH575 PLX7 Pyrimeth QS11 R33 R R RF5 Ruxolitinib SL1-1 SNS31 Sorafenib Sunitinib Temsiroli TGX1 Tipifarnib Tofacitinib TP155 Vandetanib Velaparib Vorinostat VX XV939 YM155 Zibotentan 15 5 MGH75-E MGH75-E Fig. S: Evaluation of MGH75-E using a combination drug screen.. The combination of dasatinib and ceritinib was observed as the only hit in a combination drug screen in the patient-derived cell line MGH75-E.. Dasatinib, a multi-kinase inhibitor that targets SR, was more potent in combination with 3 nm ceritinib (red) than as a single agent (black).
Figure S5 MGH7-1 eritinib-resistant, Soft Tissue iopsy D E F Fig. S5: MGH7-1 harbors an LK rearrangement.. MGH7-1 was obtained from a soft tissue lesion of a patient progressing on ceritinib. The standard, break-apart LK fluorescence in situ hybridization (FISH) assay demonstrated split signals (white arrow) in % of cells, consistent with an LK rearrangement.. positive control consisting of an independently validated lung tumor confirmed by FISH to harbor an LK rearrangement.. negative control consisting of an independently confirmed FISH negative lung cancer. D. Immunohistochemical staining for LK revealed mild membranous staining in MGH7-1. E. Positive control consisting of an independently confirmed LK-positive lung cancer assessed by immunohistochemistry. F. Negative control of an independently confirmed LK-negative lung cancer based upon immunohistochemistry.
Figure S MGH1-5 ell Viability (% control) MGH1-5 eritinib GI 5 = 73 nm 3 nm, hr Lorlatinib GI 5 = 39 nm lectinib GI 5 = 35 nm plk 1 T/Y 5 1 oncentration (nm) GPDH Fig. S: LK resistance mutations predict for sensitivity to lorlatinib in patient-derived cell line models of acquired resistance to ceritinib.. ell viability assays of the ceritinib-resistant, patient-derived cell line MGH1-5 (SQTM1-LK G1R ) treated with ceritinib, alectinib and lorlatinib. The number of cells seeded and the duration of treatment were adjusted in order to have a consistent proliferation index (3.5 to 5) at the end of treatment. Values are presented as means (N=3).. Western blot analysis of MGH1-5. ells were treated with vehicle, ceritinib (3 nm), lorlatinib (3 nm), or alectinib (3 nm) for hours. Lysates were analyzed with antibodies to the indicated proteins.
MGH-1D ell proliferation MGH51- ell proliferation Figure S7 MGH51-3 nm, hr eritinib plk 1 Lorlatinib lectinib T/Y GPDH plk 1 3 nm, hr MGH-1D D Time(d) eritinib Lorlatinib lectinib T/Y GPDH Time(d) Fig. S7: LK resistance mutations predict for sensitivity to lorlatinib in patient-derived cell line models of acquired resistance to ceritinib. -. Western blot analysis of MGH51- (EML-LK G1R ) and MGH-1D (EML-LK I1171N,115Y ), respectively. ells were treated with vehicle, ceritinib (3 nm), lorlatinib (3 nm), or alectinib (3 nm) for hours. Lysates were analyzed with antibodies to the indicated proteins. -D. ellular proliferation of MGH51- and MGH-1D cells, respectively, treated with control, ceritinib, alectinib or lorlatinib. The number of cells seeded and the duration of treatment were adjusted for each cell line in order to have a consistent proliferation index (3.5 to 5) at the end of treatment.
MGH75-E ell proliferation MGH9-1 ell proliferation Figure S 3 nm, hr MGH9-1 eritinib plk 1 Lorlatinib lectinib T/Y GPDH 3 nm, hr MGH75-E Time(d) plk 1 D eritinib Lorlatinib lectinib T/Y GPDH Time (d) Fig. S: Patient-derived cell line models of acquired resistance to ceritinib that lack LK resistance mutations are insensitive to lorlatinib. -. Western blot analysis of MGH9-1 (EML-LK WT ) and MGH75-E (EML-LK WT ), respectively. ells were treated with vehicle, ceritinib (3 nm), lorlatinib (3 nm), or alectinib (3 nm) for hours. Lysates were analyzed with antibodies to the indicated proteins. -D. ellular proliferation of MGH9-1 and MGH75-E cells treated with control, ceritinib, alectinib or lorlatinib. The number of cells seeded and the duration of treatment were adjusted for each cell line in order to have a consistent proliferation index (3.5 to 5) at the end of treatment.
MGH3- ell proliferation MGH3- ell Viability (% control) Figure S9 eritinib GI 5 = 3 nm Lorlatinib GI 5 > nm lectinib GI 5 > nm plk 1 3 nm, hr 5 T/Y 1 oncentration (nm) GPDH eritinib Lorlatinib lectinib Time (d) Fig. S9: Patient-derived cell line models of acquired resistance to ceritinib that lack LK resistance mutations are insensitive to lorlatinib.. ell viability assays of the ceritinib-resistant, patient-derived cell line MGH3- (EML-LK WT ) treated with ceritinib, alectinib and lorlatinib. Values are presented as means (N=3).. Western blot analysis of MGH3-. ells were treated with vehicle, ceritinib (3 nm), lorlatinib (3 nm), or alectinib (3 nm) for hours. Lysates were analyzed with antibodies to the indicated proteins.. ellular proliferation of MGH3- cells treated with control, ceritinib, alectinib or lorlatinib. The number of cells seeded and the duration of treatment were adjusted in order to have a consistent proliferation index (3.5 to 5) at the end of treatment.