100 osimertinib. concentration of drugs (nm) Ba/F3 C797S/del19. concentration of drugs (nm) Ba/F3 C797S/T790M/L858R. relative cell vaibility (% of
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1 Ba/F3 del19 Ba/F3 L858R a afatini afatini EGF-816 EGF-816 ty (% of ontrol) relative ell vaiilit elative ell vaiility (% of ontrol) ty (% of ontrol) relative ell vaiilit Ba/F3 T79M/L858R Ba/F3 C797S/del19 d g e f afatini afatini EGF-816 EGF-816 re g ontrol) relative ell vaiility (% of Ba/F3 C797S/T79M/del elative ell vaiility (% of ontrol) re afatini EGF-816 ontrol) 5 h relative ell vaiility (% of Ba/F3 C797S/T79M/L858R 1 1 afatini EGF-816 ty (% of ontrol) relative ell vaiili elative ell vaiility (% of ontrol) re ontrol) relative ell vaiility (% of i 5 Ba/F3 T79M/del Ba/F3 C797S/L858R afatini EGF-816 afatini EGF Ba/F3 parent 1 1 afatini erlotini Supplementary Figure 1:Ba/F3 ells expressing the ativating-mutation alone and omined with T79M, C797S and C797S/T79M, treated with linially relevant epidermal growth fator reeptor tyrosine kinase inhiitors ( TKIs) (a i) Ba/F3 ells staly expressing the ativating-mutation alone [del19 (a), L858R ()], the T79M/ativating-mutation [del19 (), L858R (d)], the C797S/ativating-mutation [del19 (e), L858R (f)], C797S/T79M/ativating-mutation [del19 (g), L858R (h)], and parent (i) were treated with the indiated onentrations of TKIs for 72 h. The CellTiter-Glo assay was used to measure ell viaility. ; =3. Results were expressed as mean ± s.d.
2 a re elative ell vaiility (% of ontro ol) rol) relative ell vaiility (% of ontr 5 5 PC9 parent (del19) PC9 triple mut (C797S/T79M/del19) afatini CO-1686 EGF afatini CO-1686 EGF-816 re elative ell vaiility (% of ontro ol) d ) relative ell vaiility (% of ontrol) 5 PC9 T79M (T79M/del19) PC9 trpl mut (C797S/T79M/del19) 1 1 onentration of Osimertini afatini CO-1686 EGF-816 -: -:1 -: -: Supplementary Figure 2: Cell growth inhiition of PC9 parent, T79M, triple-mutant ells y epidermal growth fator reeptor tyrosine kinase inhiitors ( TKIs). PC9 triple mutant treated with omination of and. (a ) PC9 parental (expressing del19) (a), PC9 T79M (expressing T79M/del19) (), PC9 triple mutant (expressing C797S/T79M/del19) () ells were treated with the indiated onentrations of first-, seond- and third-generation TKIs for 72 h. The CellTiter-Glo assay was used to measure ell viaility. (d) PC9 triple-mutant ells were treated with a omination of and. The onentrations of the onomitant were, 1,, and nm. ; =3. Results were expressed as mean ± s.d
3 relativ ve ell vaiility (% of ontrol) a 5 Ponatini 1 1 onentration of drug del19 Ponatini IC 5 T79M/del19 C797S/del19 C797S/T79M/del19 parent del T79M/del C797S/del C797S/T79M /del parent 4.9 Ponatini 3nM p C797S/ T79M/ del19 - parent + - Supplementary Figure 3: Ponatini against -triple-mutation. (a) Ba/F3 ells expressing four mutation types of -del19 were treated with the indiated onentrations of ponatini for 72 h. The CellTiter-Glo assay was used to measure ell viaility. ; =3. Results were expressed as mean ± s.d. () IC5 values were otained from same experiment. () Western lotting of Ba/F3 ells expressing the -C797S/T79M/del19 and parent indiated that treatment with 3nM of ponatini for 6 h showed no inhiitory ativity of phosphorylation of.
4 % of ontrol) relative ell vaiility ( a Ba/F3 del19 Ba/F3 T79M/del19 rigatini rigatini (% of ontrol) relative ell vaiility ( relative ell vaiility (% of ontrol) 5 Ba/F3 C797S/del19 rigatini 1 1 d IC 5 Gefitini Osimertini del T79M/del C797S/del e f g Ba/F3 del19 Ba/F3 T79M/del19 Ba/F3 C797S/del19 p p p Supplementary Figure 4: Inhiition of ell growth and signal pathway in del19, T79M/del19 and C797S/del19 mutated Ba/F3 ells. (a ) Ba/F3 ells staly expressing the -del19 alone (a), T79M/del19 () and C797S/del19 () were treated t with the indiated d onentrations ti of, i, i and i rigatini ifor 72 h. The CellTiter-Glo assay was used to measure ell viaility. ; =3. Results were expressed as mean ± s.d. (d) IC5 values were alulated using those results. (e g) Phosphorylation of and its downstream signals in Ba/F3 ells expressing -del19 alone (e), T79M/del19 (f) and C797S/del19 (g) treated y afatini,, and rigatini for 6 h were evaluated using western lotting with the indiated antiodies.
5 a ility (% of ontrol) relative ell vai relative ell vaiility (% of ontrol) 5 Ba/F3 L858R Ba/F3 T79M/L858R 1 1 rigatini rigatini e IC 5 Gefitini Osimertini L858R T79M/L858R > C797S/L858R C797S/T79M /L858R f p Afatini Osimertini L858R d vaiility (% of ontrol) relative ell ontrol) relative ell vaiility (% of 5 Ba/F3 C797S/L858R 1 1 Ba/F3 C797S/T79M/L858R 5 rigatini rigatini p p p T79M/L858R C797S/L8 858R C7 797S/T79M /L858R 1 1 Supplementary Figure 5: Inhiition of ell growth and downstream signaling in Ba/F3 ells expressing ativated y L858R treated with TKIs and rigatini (a-d) Ba/F3 ells staly expressing -L858R (a), -T79M/L858R (), -C797S/L858R () or - C797S/T79M/L858R (d) were treated with indiated onentrations of,, and rigatini for 72h. Cell viaility was assessed y CellTiter-Glo. ; =3. Results were expressed as mean ± s.d. (e) IC5 values were alulated using those results. (f) Phosphorylation of in eah mutation type of L858R introdued Ba/F3 ells treated with afatini, or rigatini was assessed using western lotting.
6 a of ontrol) Afatini A4 A549 H46 of ontrol) Osimertini A4 A549 H46 of ontrol) A4 A549 H46 relative ell vaiility (% 5 relative ell vaiility (% 5 relative ell vaiility (% onentration of drug onentration of drug onentration of drug Supplementary Figure 6: Inhiition of ell growth in - mutated ells treated with TKIs and rigatini (a ) A4 ( amplifiation), A549 (KRAS mutation) or H46 (KRAS mutation) ells were treated with the indiated onentration of afatini (a), (), and rigatini () for 72h. Cell viaility was assessed y CellTiter-Glo assay. ; =3. Results were expressed as mean ± s.d.
7 a vaiility (% of ontrol) relative ell v 5 Ba/F3 del AP26113-analogue TAE684 Ceritini ASP6 vaiility (% of ontrol) relative ell v 5 Ba/F3 T79M/del AP26113-analogue TAE684 Ceritini ASP6 relative ell vaiility (% of ontro ol) Ba/F3 C797S/del19 AP26113-analogue TAE684 Ceritini ASP d relative ell vaiility (% of ontro ol) 5 Ba/F3 C797S/T79M/del AP26113-analogue TAE684 Ceritini ASP6 e p 1 del19 AP analog 1 TAE684 1µM Ceritini 1µM ASP6 1µM f p C797S/del19 AP analog 1 1 TAE684 1µM Ceritini 1 µm ASP6 1µM Supplementary Figure 7: Effiay of rigatini and similarly strutured ALK TKIs to Ba/F3 ells expressing mutant ativated y del19. (a d) Cell growth inhiition assessed using the CellTiter-Glo assay of -mutated Ba/F3 ells [del19 alone (a), T79M/del19 (), C797S/del19 (), C797S/T79M/del19 (d)] treated with rigatini, AP analog,, TAE684, eritini, and ASP6 for 72 h. ; =3. Results were expressed as mean ± s.d. (e f) Western lotting of Ba/F3 ells expressing -del19 (E) and C797S/del19 (F) treated with the indiated drugs for 6 h showed that rigatini and AP26113-analog inhiited phosphorylation of and its downstream signaling whereas the others did.
8 a relative ell vaiility (% of o ontrol) f ontrol) relative ell vaiility (% of 5 Ba/F3 L858R Ba/F3 C797S/L858R AP26113-analogue TAE684 Ceritini ASP6 1 1 AP26113-analogue TAE684 Ceritini ASP6 ntrol) relative ell vaiility (% of o ontrol) relative ell vaiility (% of d 5 5 Ba/F3 T79M/L858R 1 1 Ba/F3 C797S/T79M/L858R 1 1 AP26113-analogue TAE684 Ceritini ASP6 AP26113-analogue TAE684 Ceritini ASP6 e AP IC 5 analog TAE684 Ceritini ASP6 L858R T79M /L858R C797S /L858R C797S/T79M /L858R Supplementary Figure 8: Effiay of rigatini and similarly strutured ALK TKIs Ba/F3 ells expressing mutant ativated y L858R. (a d) Cell growth inhiition assessed using the CellTiter-Glo assay of -mutated Ba/F3 ells [L858R alone (a), T79M/L858R (), C797S/L858R (), C797S/T79M/L858R (d)] treated with rigatini, AP26113-analog,, TAE684, eritini, and ASP6 for 72 h. ; =3. Results were expressed as mean ± s.d. (e) and AP26113-analog ahieved suffiiently low IC5 values against Ba/F3 ells expressing -C797S/T79M/L858R, otained in CellTiter-Glo assay, followed y, although other drugs were not effiient.
9 a p L858R 1 AP analog 1 TAE684 1 µm Ceritini 1 µm ASP6 1 µm p T79M/L858R 1 AP analog 1 TAE684 1 µm Ceritini 1 µm ASP6 1 µm p 1 C797S/L858R AP analog 1 TAE684 1 µm Ceritini 1 µm ASP6 1 µm d p C797S/T79M/L858R 1 AP analog 1 TAE684 1 µm Ceritini 1 µm ASP6 1 µm Supplementary Figure 9: Phosphorylation of and its downstream signaling in respetive type of -L858R mutated Ba/F3 ells treated with rigatini and similarly strutured ALK TKIs. (a-d) Western lotting of four types of -L858R mutated Ba/F3 [L858R (a), -T79M/L858R (), - C797S/L858R () or -C797S/T79M/L858R (d)] treated with the indiated ALK TKIs for 6 h showed that rigatini and AP26113-analog inhiited the phosphorylation of and its downstream signal pathway in C797S/T79M/L858R, however, TAE684, eritini, and AP6 didn t suppress the signals even at nm.
10 a Common strutured omponent O H H Cl O P O H O H S O Cl ALK-TAE684 Co-rystal -rigatini doked struture -WZ42 Co-rystal TAE684 O H WZ42 O Cl H O 5ns MD Supplementary Figure 1: Moleular doking simulation and moleular-dynamis (MD) simulation of the -C797S/T79M/L858R in omplex with rigatini. (a) Moleular doking simulation indiated that rigatini ould dok into the ATP-inding site with positional restraint on the ommon asi struture, assuming that this sustruture has a similar inding geometry etween rigatini and WZ42. Simulation algorithm was desried in the Methods. () Common strutured omponents shown in (A) of rigatini, TAE684 and WZ42 were indiated y red dashed line in eah hemial struture. () Ten distintively ategorized inding poses of rigatini into the -C797S/T79M/L858R were extrated via the doking simulation from 2 andidates and used as the initial strutures of the moleular-dynamis simulation (for 5 nse). The moleulardynamis simulation demonstrated that all ten doking modes of rigatini in ATP-inding poket onverged into one extremely similar struture as stailized onformation.
11 a p PC9 parent Afatini Osimertini AP26113-analog 1µM Gefitini 1 µm p PC9 T79M Afatini Osimertini AP26113-analog 1µM Gefitini 1 µm p PC9 parent PC9 T79M PC9 triple mutant Osimertini 1µM 1µM 1 Osimertini 1µM 1µM 1 Osimertini 1µM 1µM Supplementary Figure11: Signal pathway in PC9 ells treated with TKIs, rigatini and. (a ) Phosphorylation of and its downstream signal pathway in PC9-parent (del19) (a) and -T79M (T79M/del19) () ells treated with indiated TKIs were evaluated using western lotting with indiated antiody. Afatini and showed signifiantly inhiit the signals of oth types of PC9 ells while rigatini and AP26113-analog moderately do. () Western lotting of PC9 parent, -T79M, -triple mutant (C797S/T79M/del19) ells treated with, rigatini, and demonstrated that rigatini and sustantially ahieved the inhiition of phosphorylation of and its downstream signal pathway in PC9-triple mutant ells.
12 a PC9-triple mutant (C797S/T79M/del19) Vehile Osimetini Tumor volume (mm3) Vehile Control Osimertini ** * * * Days after initial treatment p Body weight hange (g) Vehile Control Osimertini * * * * Days after initial treatment Supplementary Figure 12: ut not suppressed the growth of - C797S/T79M/del19 expressing PC9 ells in vivo. (a ) PC9 ells expressing -C797S/T79M/del19 were suutaneously implanted into Bal- nu/nu mie. When the average tumor volume reahed approximately 2 mm 3, the mie were randomized into vehile ontrol or treatment groups (5 mg kg -1 of or 75 mg kg -1 of rigatini, respetively) and treated one daily y oral gavage for the indiated period. Tumor volume (V) was alulated as.5 length width 2, and ody weights (B.W.) of mie were measured twie weekly. ; =6. Results in a and are expressed as mean ± s.d. ; **P <.1,*P <.5 (Mann Whitney U test). () Phosphorylation of and its downstream signaling in two tumor samples otained from eah group were evaluated using western lotting.
13 a Tumor volume (m mm 3 ) 1, Perent survival PC9-T79M (T79M/del19) ** ** Vehile Control Osimertini Days after initial treatment Survival proportions of PC9-T79M (g) Body Weight ( d Vehile Osimertini PC9-T79M Vehile Control Osimertini Days after initial treatment p PC9-T79M (T79M/del19) Day after initial treatment Supplementary Figure 13: with etuxima inhiited the tumor growth of - T79M/exon19 deletion mutation haroring PC9 ells in vivo. (a ) Gefitini resistant PC9 ells expressing -T79M/del19 (PC9-T79M) were suutaneously implanted into Bal- nu/nu mie. When the average tumor volume reahed approximately 2 mm 3,the mie were randomized into vehile ontrol or treatment groups (5 mg kg -1 of or 75 mg kg -1 of rigatini, respetively) and treated one daily y oral gavage for the indiated period. Tumor volume (V) was alulated as.5 length width 2, and ody weights (B.W.) of mie were measured twie weekly. ; =6. Results in a and areexpressedasmean± s.d. ; **P <.1 (Mann Whitney U test).() Survival proportion p desried using the Kaplan Meier method showed and rigatini prolonged the survival of mie into whom PC9-T79M xenogfrafts were implanted. One rigatini treated mie was aidentally killed y the proedure of oral gavage at day 1. (d) Bal- nu/nu mie earing the PC9-T79M ells treated with vehile ontrol or treatment groups (75 mg kg -1 of rigatini or 75 mg kg -1 of rigatini with 1 mg per mouse of etuxima, respetively). was treated one daily y oral gavage, and etuxima was treated 3 times/week. After 1 days of the treatment, phosphorylation of and its downstream signaling in two tumor samples otained from eah group were evaluated using western lotting.
14 a relative ell viaility (% of on ntrol) Ba/F3 C797S/T79M/del19 ** nm /pani(-) TKI/pani(-) /pani(+) TKI/pani(+) ** n.s. AP26113-analog nm Osimeritini 3nM relativ ve ell viaility (% of ontrol) PC9-triple mutant MGH121 res2 /pani(-) TKI/pani(-) /pani(-) TKI/pani(-) /pani(+) TKI/pani(+) /pani(+) TKI/pani(+) ** n.s. ** ** * nM Osimertini 3nM ive ell viaility (% of ontrol) relat 2 3nM Osimertini 3nM Supplementary Figure 14: Comination with panitumuma enhaned the poteny of rigatini (a) The ell growth inhiition of Ba/F3 ells expressing -C797S/T79M/del19 (-triple-del19) treated with rigatini, AP26113-analog and at indiated onentrations omined with or without panitumuma (2 µg/ml) for 72 h assessed using CellTiter-Glo assay. ( ) The ell growth inhiition of PC9-triple mutant ells () or MGH121 res2 ells (), oth expressing -triple-del19, treated with rigatini and at indiated onentrations omined with or without panitumuma (2 µg/ml) for 72 h assessed using CellTiter-Glo assay. Results are expressed as mean ± s.d. =3. ; n.s.: not signifiant, **P <.1, *P <.5 (Student s t-test).
15 Supplementary Figure 15: Original immunolots for indiated figures Figure 1 Afatini Osimer -tini p 1 1 1
16 Supplementary Figure 16: Original immunolots for indiated figures Figure 3d,e T79M/del19 C797S/T79M/del19 p n 1 AP analog 1 E684 1 µm ritini 1 µm P6 1 µm TAE Cer ASP n 1 AP analog 1 E684 1 µm ritini 1 µm P6 1 µm TAE Cer ASP
17 Supplementary Figure 17: Original immunolots for indiated figures Figure 5f p Afatini Osimer tini AP analog 1uM Gefiti ini 1uM Figure 5g Afatini Osimer tini p AP26113-analog 1uM Gefitini 1uM 1
18 Supplementary Figure 18: Original immunolots for indiated figures Figure Cetuxima p Figure 6d Cetuxima p
19 Supplementary Figure 19: Original immunolots for indiated figures Figure 6f Cetuxima p
20 Supplementary Figure 2: Original immunolots for indiated figures Figure 7 p 6 h Cet. 1 µg/ml Brig. 5 nm h 48 h Brig. + et. Cet. 1 µg/ml Brig. 5 nm Brig. + et. Cet. 1 µg/ml Brig. 5 nm Brig. + et. Figure 7d p 6 h on et. 1 µg/ml rig. 5 nm rig. + et. et. 1 µg/ml h 48 h rig. 5 nm rig. + et. et. 1 µg/ml rig. 5 nm rig. + et. no Ce Br Br Ce Br Br Ce Br Br 17 Bim 17 Bim 17
21 Supplementary Figure 21: Original immunolots for indiated figures Figure 8d p Figure 8h p Bim 17 17
22 Drug Company Drug Company 1 17-AAG LC laoratories 17 EGF-816 ChemSene 2 AEW541 AtiveBiohem 18 Erlotini LC laoratories 3 Afatini ChemieTek 19 Foretini AdooQ Biosiene 4 Aletini AtiveBiohem 2 Gefitini LC laoratories 5 AP26113-analogue Sellek 21 Imatini LC laoratories 6 ASP6 ChemieTek 22 Lapatini LC laoratories 7 BioVision 23 Lorlatini AtiveBiohem 8 BGJ398 Shanghai Biohem intedani Sellek 9 Shanghai Biohem 25 Osimertini Sellek 1 Caozantini AtiveBiohem 26 PHA6657 Toris Biosiene 11 CEP71 Caliohem 27 Ponatini Sellek 12 Ceritini AtiveBiohem 28 Sorafeni Sellek 13 CO-1686 AtiveBiohem 29 Sunitini Sellek 14 Crizotini Biohempartner 3 TAE684 ChemieTek 15 E78 Sellek Vandetani Shanghai Biohem 16 Cetuxima Merk Panitumuma Takeda Pharma. Supplementary Tale 1: Kinase inhiitors and other drugs The drugs used in the experiments and the ompanies from whih they were purhased are shown in tale.
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