Supplementary Information. Title: RAS-MAPK dependence underlies a rational polytherapy strategy in EML4-

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1 Supplementry Informtion Title: RAS-MAPK dependence underlies rtionl polytherpy strtegy in EML4- ALK positive lung cncer Authors: Gorjn Hrustnovic, Victor Olivs, Evngelos Pzrentzos, Asmin Tulpule, Surh Asthn, Collin M. Blkely, Ross A. Okimoto, Luping Lin, Dn S. Neel, Amit Snis, Jennifer Flngn, Elton Chn, Mrileil Vrell-Grci, Dr L. Aisner, Ari Vishnvi, Si-Hong I. Ou, Eric A. Collisson, Eiki Ichihr, Philip C. Mck, Christine M. Lovly, Niki Krchliou, Rfel Rosell, Jonthn W. Riess, Roert C. Doeele, Trever G. Bivon Supplementl Figures nd Legends.

2 Drug 24h: D SO M C i in ni M ot iti M nm n ri z er 1u C 1 1 Selumetini 5 n M 1u M ni iti 22 9 M12 L71 ol C K ux D R BY M BK G DM S Tr O me tini Ru xo l itin i GD C 94 1 BK M1 2 BY L7 19 Trmetini DM S Tr O me tini Ru xo l itin i GD C 94 1 BK M1 2 BY L7 19 DM S Tr O me tini Ru xo l itin i GD C 94 1 BK M1 2 BY L7 19 Figure S1 tstat3 / GAPDH GAPDH 6h 48h 24h c Trmetini MEK162 PD32 1 nm.36 1 nm 8 e Drug (nm): d 1 nm Trmetini 1 MEK162 PD nm 1 nm 7.38 pbabe,ev" Criz. 1uM: """"m"""""""3m"""""6h" 1 nm "BRAF"V6E" EML4 tstat3 V6E GAPDH GAPDH g 1 5 pb A B EpB EV A B EB R A F V6 E Crizotini: 1 nm 25 nm pbabeev Crizotini IC5 (nm) pbabebraf V6E f 5 nm 75 nm 1 nm

3 Figure S1. RAS-MAPK signling regultes oncogene dependence in EML4-ALK lung denocrcinom cells. () Immunolot nlysis using indicted ntiodies in cells treted with inhiitors (24h) from Fig. 1c. Trget inhiition t concentrtions used is shown. () Immunolot nlysis using the indicted ntiodies in cells treted with indicted inhiitors t concentrtions from Fig. 1c for 6h, 24h, or 48h, indicting durtion of trget inhiition up to 48h (t the time of medi chnge for the growth ssy in Fig. 1c). (c-d) cells were treted with severl different MEK inhiitors (trmetini, MEK162, PD32) t the indicted concentrtions nd (c) viility ws mesured over 5-dy crystl violet ssy. (d) Immunolot nlysis of cells with the indicted ntiodies treted with the indicted MEK inhiitors for 24h. (e-g) cells were trnsduced with BRAF V6E nd then (e) immunolot nlysis ws performed on cells treted with 3m or 6h crizotini (1uM), (f) IC5 vlues were estimted from crizotini dose response (cell titer glo), nd (g) cells were plted nd sujected to 5-dy growth ssy in the presence of indicted concentrtions of drug, then fixed nd stined with crystl violet. All dt shown represent t lest 3 independent experiments.

Figure S2 Crizotini (nm) 25 5 c 1 KRAS -G12C GFP 6h Crizotini (5nM): GFP KRAS -G12D KRAS -G12V CA.STAT3 GFP 24h$Crizo*ni$(5$nM):$.35.49.14 talk G12C talk.86.91 FLAG.8 KRAS tstat3 G12D.95.

STAT3 g GFP 6h Crizotini (5nM): KRAS -G12C KRAS -G12D KRAS -G12V h talk talk tstat3 FLAG RAS GAPDH GAPDH STE-1 CA.STAT3 GFP 24h Crizotini (5 nm): STE-1 T3.24 5 Crizotini (nm) TA.22 25.S.32 A C 9 D.

4 Figure S2 Crizotini (nm) 25 5 c 1 KRAS -G12C GFP 6h Crizotini (5nM): GFP KRAS -G12D KRAS -G12V CA.STAT3 GFP 24h$Crizo*ni$(5$nM):$ talk G12C talk FLAG.8 KRAS tstat3 G12D G12V GAPDH RAS CA.STAT3 2 d Crizotini (nm) 5 e Ceritini (nm) 1 5 f 2 STE1 1uM Crizotini 2 S- R A R K K V.14 1 Construct CA.STAT3 g GFP 6h Crizotini (5nM): KRAS -G12C KRAS -G12D KRAS -G12V h talk talk tstat3 FLAG RAS GAPDH GAPDH STE-1 CA.STAT3 GFP 24h Crizotini (5 nm): STE-1 T Crizotini (nm) TA S.32 A C 9 D G.78 G12V.2 S- A 8 C G.75 S- G12D P <.1.4 A.38 R.67 K.44 FP.71 P <.1 P <.1.6 G GFP KRAS-G12C KRAS-G12D KRAS-G12V CA.STAT3 G KRAS G12C Reltive Viility (72hr) STE-1.8 Frction Viility (1uM Criz/) GFP

5 Figure S2. Activtion of RAS signling promotes resistnce to ALK inhiitors vi rescue of MAPK signling. (-c) cells expressing the indicted cdnas (GFP, KRAS G12C/D/V), or CA.STAT3) were ssyed for () viility in the presence of crizotini nd (-c) sujected to immunolot nlysis with the indicted ntiodies nd drug tretments. (d-h) STE-1 cells expressing the indicted cdnas (GFP, KRAS G12C/D/V), or CA.STAT3) were ssyed for (d) viility in the presence of crizotini using either (d) crystl violet ssy or (e-f) 72h Cell Titer Glo ssy. These sme cells were then sujected to (g-h) immunolot nlysis with indicted ntiodies nd indicted tretments. All dt shown represent t lest 3 independent experiments.

6 Figure S3 EGFR Reltive Viility/ H165 Reltive Viility/ HCC827 Reltive Viility/ H1975 Reltive Viility/ H3255 Reltive Viility/ 1118 H23 H358 A549 H23 CALU-6 KRAS Reltive Viility/ Reltive Viility/ Reltive Viility/ Reltive Viility/ Reltive Viility/ CAL-12T HCC364 H1395 H245 H1666 H287 BRAF Reltive Viility/ Reltive Viility/ Reltive Viility/ Reltive Viility/ Reltive Viility/ Reltive Viility/ STE-1 ALK Reltive Viility/ Reltive Viility/ Cell Line Oncogene' Mut,on' Trme,ni' IC5' H165' EGFR' Del19' >1uM' HCC827' EGFR' Del19' >1uM' H1975' EGFR' L858R,T79M' >1uM' H3255' EGFR' L858R' >1uM' ROS1 Reltive Viility/ HCC ' EGFR' L858R' >1uM' H23' KRAS' G12C' 85nM' H358' KRAS' G12C' 65nM' A549' KRAS' G12S' 3nM' H23' KRAS' G12C' >1uM' CALUL6' KRAS' Q61K' 25nM' CALL12T' BRAF' G466V' 15nM' Lung Epithelium Reltive Viility/ Bes2B HCC364' BRAF' V6E' 5nM' H1395' BRAF' G469A' 5nM' H245' BRAF' L485Y' 125nM' H1666' BRAF' G466V' 25nM' H287' BRAF' L597V' 5nM' ' EML4LALK' E13:A2' 25nM' STEL1' EML4LALK' E13:A2' 7nM' HCC78' SLC34A2LROS1' S12:R32' >1uM' Bes2B' N/A' N/A' >1uM'

7 Figure S3. MEK dependence in EML4-ALK nd other lung denocrcinom cell lines. () The indicted cell lines (ll ptient-derived lung cncer lines, except Bes2B which re immortlized humn ronchil epithelil cells lcking driver oncogene) were treted with incresing doses of trmetini (,,, nd ) for 72h nd viility ws mesured using cell titer glo. () Tle showing the indicted cell lines tested nd their respective genetic drivers. IC5 vlues were estimted from growth curves in (). (n>3, dt re shown SEM for quntittive ssys ). All dt shown represent t lest 3 independent experiments.

8 Figure S4 ) ) ) sih$ras:) 25)nM) ) sin$ras:) ) 25)nM) ) ) ) ) sik$ras:) 25)nM) Ceri/ni:) ) ) ) ) ) siscr.:) 75)nM) 5)nM) 5)nM) 5)nM) ) 25)nM) 25)nM) 25)nM) EML4/ALK$ Vrint$3$$ (E6;A2)$ EML4$ Bsic$ Kinse$ ALK$ Rs GAPDH Crizotini: IP:GST-RBD Whole cell lyste!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! H2228 Rs Rs Actin STE-1 c d e f nm Drug Crizotini IC5 Dose Drug Ceritini H2228 Reltive Viility (72hr) g Reltive Viility/ Crizotini (um) H H2228 Reltive Viility (72hr) Ceritini (um) H

9 Figure S4. EML4-ALK engges H-, N-, K-RAS vi the EML4 HELP domin. () Immunolot nlysis with indicted ntiodies on STE-1 cell lystes trnsfected with indicted sirnas t the indicted concentrtions for 72h. () Grphicl depiction of EML4-ALK vrint 3 present in H2228 lung denocrcinom cells nd tht lcks the HELP domin in EML4, in contrst to EML4-ALK vrint 1 expressed in lung denocrcinom cells tht contins the HELP domin in EML4. (c) Immunolot nlysis in H2228 cells (whole cell lyste or GST-RBD IP, s indicted) with the indicted ntiodies. H2228 cells were treted with 5nM Crizotini for 1hr. (d-e) Cell titer glo viility ssys in nd H2228 cells treted with the indicted drug (d) crizotini or (e) ceritini) for 72hr. The r grph (f) depicts IC5 estimtion derived from the dt. (n>3, dt re shown SEM for quntittive ssys nd for immunolots nd immunofluorescence representtive of >3 independent experiments). (g) Response of H2228 cell line to trmetini monotherpy using 72h Cell Titer Glo ssy. All dt shown represent t lest 3 independent experiments.

10 Figure S5 4 c Erlotini: Trmetini: pegfr tegfr 2 1 Selumetini (1uM) SCH (1uM) Alectini () AlSel AlSCH Actin H3255 d Crizotini: Trmetini: talk Crizotini: Trmetini: talk e Reltive Viility (72hr) Trmetini Trmetini cparp Actin cparp Actin STE Crizotini (um) f Crizotini (5 nm): Trmetini (1 nm): talk tstat3 cparp GAPDH H2228

11 Figure S5. Effects of MAPK pthwy inhiition in the lung denocrcinom models. () Effect of indicted drug tretments on cell growth. Cells were plted in 6-well pltes nd cell numer ws determined 7d fter tretment. Vlues re presented s the percent of cells remining on d7 compred to vehicle tretment. () H3255 cells (EGFR L858R lung denocrcinom) were treted with erlotini for 72h with either or trmetini in ddition. Viility ws mesured using cell titer glo nd vlues were normlized to / control. (c) Immunolot nlysis in H3255 cell lystes following tretment for 1h with either erlotini (), trmetini (), or the comintion. (d) Immunolot nlysis in nd STE-1 cell lines treted with the indicted drugs (higher dose of Trmetini thn Fig. 3d) for 24h. (e) H2228 cells treted with su-mximl doses of trmetini (nd comintion with Crizotini) s shown in Fig. 3- using 72h Cell Titer Glo ssy to ssess viility. (f) Immunolot nlysis using indicted ntiodies on H2228 cells treted with indicted drugs for 24h.

12 Figure S6 Xenogrft 1 Tumor Volume mm Vehicle (n=6) Ceritini 25mg/kg (n=8) Trmetini 3mg/kg (n=8) Ceritini 25mg/kg Trmetini 1mg/kg (n=8) Avg. Volume d= % loss in ody weight t = d % Body Weight Loss (g) Xenogrfts P = P =.16 P =.86 Vehicle Trmetini 3 mg/kg Ceritini 25 mg/kg Ceritini 25 mg/kg Trmetini 1 mg/kg c % Body Weight Chnge % Body Weight Loss (g) STE-1 Xenogrfts Vehicle Trmetini 1mg/kg Crizotini 5mg/kg Crizotini 5mg/kg Trmetini 1mg/kg -25 le kg kg g g -2 e g g d 5 nm Ceritini 1uM Ruxolitini JAK Inhiitors 1uM 1uM Bricitini Momlotini 1uM Tofecitini e tstat3 JAK inhiitor (1 um) Bricitini Tofecitini Momlitini 1 um Ruxolitini 1 um Bricitini 1 um Momlotini 1 um Tofecitini STE-1 5 nm Ceritini GAPDH f Tumor Volume mm Xenogrft P <.1 P <.1 Vehicle (n=6) Ruxolitini 2mg/kg (n=1) Trmetini 1mg/kg (n=1) Ceritini 25mg/kg (n=1) Ceritini 25mg/kg Ruxolitini 2mg/kg (n=1) Ceritini 25mg/kg Trmetini 1mg/kg (n=1) Dy

13 Figure S6. Effects of dul ALK nd su-mximl MEK inhiition in the in vivo EML4-ALK lung denocrcinom models. () xenogrfts from Fig. 3e. Mice were treted with indicted concentrtions of drug once dily. (-c) Percent loss in ody weight (g) of nude mice engrfted with () or (c) STE-1 cells nd treted with indicted regimens. Vlues represent chnge in weight from study endpoint (d=23 or d=31) from seline (d=). P vlue ws determined using unpired t-test etween tretment group nd vehicle. (n>3, dt re shown SEM for quntittive ssys nd for immunolots representtive of >3 independent experiments). (d) Crystl violet cell growth ssys in the indicted cells treted with the indicted gents, including JAK inhiitors s shown. All dt shown represent t lest 3 independent experiments. (e) Immunolot nlysis of cells treted with JAK inhiitors (24h) from (d). (f) Nude mice engrfted with cells were treted with indicted tretment regimens for 13d, nd tumor size (mm 3 ) ws mesured.

Figure S7 % sensi-ve% [crizo'ni] resistnt% CAR1% CAR2% CAR3% Cell Line Crizotini IC5 (nm) Ceritini IC5 (nm) Growth Rte/ 22 11 ±.2 CAR1 28 15 1.29 ±.6 CAR2 42 16.79 ±.4 CAR3 41 155.75 ±.

14 Figure S7 % sensi-ve% [crizo'ni] resistnt% CAR1% CAR2% CAR3% Cell Line Crizotini IC5 (nm) Ceritini IC5 (nm) Growth Rte/ ±.2 CAR ±.6 CAR ±.4 CAR ±.6 % sensi-ve% [crizo'ni] LDK378' resistnt% CAR1% LAR1% CAR2% LAR2% CAR3% LAR3% LAR1 21 >2.95 ±.1 LAR ±.6 LAR3 16 >2 1.2 ±.16 c Inhiitor ALK ALK MEK AKT PI3K PI3K JAK LAR1 LAR2 Crizotini Ceritini Trmetini MK226 BKM12 BYL719 Ruxolitini

15 Figure S7. ALK inhiitor resistnt EML4-ALK lung denocrcinom cell lines derived nd studied. () Schemtic representtion of the protocol to derive ALK TKI isogenic resistnt cell lines. were treted with esclting doses of crizotini (, 25nM, 5nM, ) or ceritini (, 5nM,, 2nM) for 9 dys. Resistnt lines were mintined t either 1uM crizotini (CAR) or 2nM ceritini (LAR). () Crizotini nd ceritini IC5 vlues for CAR nd LAR lines. Growth rtes of CAR nd LAR lines (in the presence of either 1uM crizotini or 2nM ceritini, respectively) re depicted reltive to prentl growth rte. (c) LAR1 nd LAR2 cells were plted in 6-well pltes nd treted with indicted concentrtion of indicted inhiitors. Cells were fixed t dy 5 nd stined with crystl violet. ). (n>3, dt re shown SEM for quntittive ssys nd for crystl violoet ssys representtive of >3 independent experiments)

Figure S8 KRAS log2 copy numer rtio 3 2 1 Trnscript Aundnce 5 4 3 2 1 KRAS-4A KRAS-4B % CAR1% PCR% KRAS%4B%(57p)% GAPDH% -1 2 3 4 CAR1 Chromosome 12 (M) c d e shscr shkras1 shkras2 KRAS cparp (89kd)

Amplifiction d 3 49 F Crizotini ALK L1196M None Dupliction c 4 42 F Crizotini ALK CNG None Dupliction c 5 4 M Crizotini, AP26113 None Not tested Dupliction c 6 54 M Crizotini None None Dupliction c 7

Negtive 12 51 M Crizotini, AP26113 ALK CNG None Negtive 13 53 F Crizotini None None e Amplifiction d 14 41 F Crizotini None None e Amplifiction d 15 44 M Crizotini None None Dupliction c KRAS, NRAS,

16 Figure S8 KRAS log2 copy numer rtio Trnscript Aundnce KRAS-4A KRAS-4B % CAR1% PCR% KRAS%4B%(57p)% GAPDH% CAR1 Chromosome 12 (M) c d e shscr shkras1 shkras2 KRAS cparp (89kd) ccasp3 (17,19kd) Actin CAR1 f Ptient Age Sex ALK inhiitor ALK resistnce muttion RAS/RAF/MEK muttion KRAS Copy Numer Altertion 1 27 F Ceritini ALK F1174C None Negtive 2 7 M Crizotini None None Amplifiction d 3 49 F Crizotini ALK L1196M None Dupliction c 4 42 F Crizotini ALK CNG None Dupliction c 5 4 M Crizotini, AP26113 None Not tested Dupliction c 6 54 M Crizotini None None Dupliction c 7 62 F Crizotini ALK G1269A None Dupliction c 8 41 M Crizotini None None Negtive 9 41 F Crizotini None None Negtive 1 75 M Crizotini EGFRdel19 None Dupliction c F Crizotini, Ceritini None None Negtive M Crizotini, AP26113 ALK CNG None Negtive F Crizotini None None e Amplifiction d F Crizotini None None e Amplifiction d M Crizotini None None Dupliction c KRAS, NRAS, BRAF, MEK y SNPshot y direct sequencing for KRAS, NRAS, BRAF, MEK1/2 c typicl KRAS FISH pttern including doulets in post-tretment iopsy d KRAS/CEP12 rtio > 2.2 in post-tretment tumor cells e y direct sequencing for NRAS, BRAF, MEK1/2

17 Figure S8. KRAS WT genetic upregultion drives ALK inhiitor resistnce. () Exome nlysis of CAR1 revels focl mplifiction on chromosome 12 spnning KRAS WT. () qpcr using primers designed for KRAS-4A nd KRAS-4B trnscripts in nd CAR1. Inset shows representtive semi-quntittive PCR results. (c) Growth inhiition in response to KRAS sirna trnsfection /- crizotini in CAR1. Cells were trnsfected for 48h, nd treted with indicted concentrtion of crizotini for 96h, then fixed nd stined with crystl violet. (d) CAR1 cells were trnsduced with two independent shrnas trgeting KRAS. Cells were then plted nd exposed to the indicted doses of crizotini nd fixed nd stined t 5 dys fter tretment. (e) Immunolot nlysis with the indicted ntiodies in CAR1 cell lystes fter introduction of either shrna-scrmle or shkras1, or shkras2. (f) Tle summrizing ALK fusion lung denocrcinom ptient chrcteristics (n=15). All ptients were treted with the indicted ALK TKI nd tumor iopsies were otined fter resistnce (where ville, the corresponding pretretment tumor smple ws lso nlyzed, ptients #2, #13, #14). The presence of ALK, RAS, RAF, or other muttions re noted long with KRAS copy numer ltertion sttus (where sufficient tumor smple ws ville for such nlysis).

18 Figure S GFP 3 KRAS WT (Virus Titrtion) KRAS m.o.i.: Criz 5nm (6h): RAS HSP9 GFP c Post-crizotini resistnce Ptient 6

19 Figure S9. Effects of reltively low levels of KRAS WT expression in EML4-ALK lung denocrcinom cell lines. (-) cells were trnsduced with incresing virl titrtions to chieve differentil levels of KRAS WT expression. These isogenic cells were then ssyed for Crizotini response using () Crystl Violet ssy nd () immunolot nlysis with the indicted ntiodies to ssess downstrem signling (immunolots nd crystl violet ssys representtive of >3 independent experiments). (c) Representtive imge of KRAS gene dupliction events s mesured y KRAS FISH in ALK fusion positive ptient tumor iopsy with cquired resistnce to ALK TKI (the ALK inhiitor resistnt tumor specimen from ptient #6 is shown). White rrowheds indicte tumor cells with KRAS copy numer gin events.

Figure S1 Crizo2ni:( GST$RBD( ( CAR1( CAR2( CAR3( ( ( ( ( ( ( ( ( Phosphtse DUSP28 DUSP23 DUSP22 DUSP19 DUSP18 DUSP16 DUSP14 DUSP13 DUSP12 DUSP11 DUSP1 DUSP9 DUSP8 DUSP Expression CAR2 CAR3 DUSP7

20 Figure S1 Crizo2ni:( GST$RBD( ( CAR1( CAR2( CAR3( ( ( ( ( ( ( ( ( Phosphtse DUSP28 DUSP23 DUSP22 DUSP19 DUSP18 DUSP16 DUSP14 DUSP13 DUSP12 DUSP11 DUSP1 DUSP9 DUSP8 DUSP Expression CAR2 CAR3 DUSP7 WCL( DUSP6 DUSP5 DUSP4 DUSP3 DUSP2 DUSP Expression (FPKM) 4 5 c Ptient Age Sex ALK inhiitor DUSP6 IHC Score (-4) Pre-Tretment Post-Crizotini Resistnce Post-Crizotini Tretment, Response F Crizotini 4 2 ASP326, 4m F Crizotini 3 2 ASP326, 4m M Crizotini 2 c ASP326, 4.5m 16 4 M Crizotini 3 1 d MPDL328A, 16m F Crizotini n 2 d n F Crizotini n 2 d Ceritini, 8m 19 3 M Crizotini 2 2 d n 2 6 F Crizotini n Ceritini, 2m M Crizotini 3 1 d Chemotherpy, 4m F Crizotini 2 n n M Crizotini 1 n n M Crizotini 2 n n F Crizotini 3 n n M Crizotini 2 n n F Crizotini 4 n n F Crizotini 1 n n F Crizotini 2 n n 3 45 M Crizotini n No ALK TKI 31 6 M Crizotini 3 n n = mtched smples = ALK TKI (tretment post-crizotini) Cropltin/Pemetrexed KRAS CNG positive KRAS CNG negtive c KRAS doulets/dupliction d KRAS CNG not tested d

21 Figure S1. RAS-MAPK pthwy ctivtion in ALK inhiitor resistnt EML4- ALK lung denocrcinom. () GST-RBD pull down ssys in nd CAR lines showing RAS-GTP levels -/ 5nM crizotini (6hr). () mrna expression levels (fpkm) of DUSP fmily memers in, CAR2, nd CAR3. (c) Tle showing the ptient cohort ssyed for DUSP6 using IHC (score -4). Mtched pirs re indicted with lue sterisk. n indictes no ville tissue for nlysis. Additionlly, post- Crizotini tretments re indicted where ville. (d) Exmples of DUSP6 IHC scores used in (c). A score of represents no pprent DUSP6 stining in tumor cells, 1 represents low levels of identifile stining in ny tumor cell popultions. 2 represents moderte DUSP6 stining intensity in tumor cells. 3 represents high, nd 4 represents very high DUSP6 stining in tumor cells. Scoring ws performed y 3 linded individuls nd consensus scores re presented.

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION doi:1.138/nture1794 BR EPFs BRI1? ERECTA TMM BSKs YDA PP2A BSU1 BIN2 pbzr1/2 BZR1/2 MKK4/5/7/9 MPK3/6 SPCH Cell growth Stomtl production Supplementry Figure 1. The model of BR nd stomtl signling pthwys.