Overcoming EGFR T790M-based Tyrosine Kinase Inhibitor Resistance with an Allele-specific DNAzyme

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1 Cittion: Moleculr Therpy Nucleic Acids (214) 3, e15; doi:1.138/mtn The Americn Society of Gene & Cell Therpy All rights reserved /14 Overcoming T79M-sed Tyrosine Kinse Inhiitor Resistnce with n Allele-specific DNAzyme Wei-Yun Li 1 3, Chi-Yun Chen 4, Shuenn-Chen Yng 3, Jer-Yurn Wu 3, Cheng-Ju Chng 3, Pn-Chyr Yng 3,5 nd Konn Peck 3,6 Epiderml growth fctor receptor () tyrosine kinse inhiitors (TKIs) re the min therpeutic gents used to tret non smll-cell lung cncer ptients hroring -ctivting muttions. However, most of these ptients will eventully develop resistnce, 5% of which re due to secondry muttion t T79M in the. In this pper, we descrie the development of n llele-specific DNAzyme,, tht cn specificlly silence T79M mutnt messenger RNA while leving wild-type intct. Allele-specific silencing of T79M expression nd downstrem signling y triggered poptosis in non smll-cell lung cncer cells hroring this mutnt. Adding cholesterol-triethylene glycol group on the 3 -end of (c) improved drug efficcy, incresing inhiitory effect on cell viility from 46 to 79% in T79M/L858R-hroring non smll-cell lung cncer cells, without loss of llele specificity. Comined tretment with c nd BIBW-2992, second-genertion -tyrosine kinse inhiitor, synergisticlly inhiited downstrem signling nd suppressed the growth of xenogrft tumors derived from cells. Collectively, these results indicte tht the llele-specific DNAzyme,, my provide n lterntive tretment for non smll-cell lung cncer tht is cple of overcoming T79M mutntsed tyrosine kinse inhiitor resistnce. Moleculr Therpy Nucleic Acids (214) 3, e15; doi:1.138/mtn.214.3; dvnce online puliction 4 Mrch 214 Introduction Non smll-cell lung cncer (NSCLC) is the leding cuse of cncer-relted deth. 1,2 In one unique suset of NSCLC ptients, lung cncer cells hror ctivting muttions in the epiderml growth fctor receptor () nd ecome ddicted to errnt signling for their survivl. 3,4 Among ctivting muttions, L858R nd exon 19 deletion in ccount for over 9% of drug-sensitive muttions nd show incresed inding ffinity towrd tyrosine kinse inhiitors (TKIs) compred to the wild-type. 5,6 The dministrtion of TKIs successfully induces intrinsic poptosis pthwys in -mutnt lung cncer cells; however, doselimiting side effects re lso unvoidly triggered y the concurrent inhiition of wild-type signling in norml cells. 7,8 Moreover, despite the success of TKIs t the eginning of NSCLC tretment, the cquired secondry muttion t the gtekeeper residue 79 of (T79M), which is found in 5% of drug-resistnt ptients, wekens the interction etween TKIs nd Dose-limiting toxicity nd T79M-sed drug resistnce re the min issues in NSCLC tretment tht still remin to e solved. DNAzymes DNA oligonucleotides with ctlytic ctivity towrd specific trget RNA sequences hve een comprehensively studied s mens for silencing vrious genes nd hve shown promise for use s therpeutic gents The DNAzymes 1 23 nd 8 17 hve een most ctively studied. DNAzymes consist of conserved ctlytic core tht is essentil for ctlytic ctivity, flnked y two sustrte-inding rms with sequences complementry to the trgeted messenger RNA (mrna) sequence. 15 A single mismtch in the inding rm cn impede the enzymtic ctivity of the DNAzyme; therefore, with rtionl design, DNAzymes re cple of distinguishing etween mutnt nd wild-type mrnas nd chieving specific clevge. 16,17 It hs een shown tht DNAzyme ginst the K-Rs (G12V) mutnt form specificlly cleves K-Rs mutnt mrna nd leves wild-type K-Rs mrna intct. 18 Also, c-jun mrna-trgeting DNAzyme ws shown to suppress the growth of skin cncers (oth sl cell nd squmous cell crcinoms) in mouse xenogrft models In preclinicl studies, DNAzymes were shown to e sfe nd well tolerted, nd exhiited cceptle off-trget effects. 21 In this study, we developed n llele-specific DNAzyme () ginst T79M nd showed tht it effectively inhiited T79M expression nd suppressed xenogrft tumor growth. Comined therpy with nd BIBW synergisticlly inhiited the growth of xenogrft tumors derived from cells hroring the T79M mutnt, suggesting tht my overcome T79M-sed TKI resistnce in NSCLC. Results Design of DNAzymes (Figure 1) ws designed ccording to the prototype of the 1 23 DNAzyme, which comprises 15-deoxyrionucleotide ctlytic core 22 nd flnking side rms, I nd II, with sequences complementry to its RNA trget. The gene encoding T79M hs C to U sustitution t the T79M muttion site. 1 Severl DNAzymes ginst T79M were tested for their efficcies in discriminting T79M The lst three uthors contriuted eqully to this work. 1 Tiwn Interntionl Grdute Progrm in Moleculr Medicine, Ntionl Yng-Ming University nd Acdemi Sinic, Tipei, Tiwn; 2 Institute of Biochemistry nd Moleculr Biology, Ntionl Yng-Ming University, Tipei, Tiwn; 3 Institute of Biomedicl Sciences, Acdemi Sinic, Tipei, Tiwn; 4 Deprtment of Nutrition nd Helth Sciences, Chng Gung University of Science nd Technology, Toyun, Tiwn; 5 College of Medicine, Ntionl Tiwn University, Tipei, Tiwn; 6 Author decesed. Correspondence: Pn-Chyr Yng, President of Ntionl Tiwn University, 1, Sec. 4, Roosevelt Rd., Tipei 16, Tiwn. E-mil: pcyng@ntu.edu.tw. keywords: DNAzyme; T79M; NSCLC; TKI Received 9 Octoer 213; ccepted 3 Jnury 214; dvnce online puliction 4 Mrch 214. doi:1.138/mtn.214.3

2 2 Overcoming -TKI Resistnce with DNAzyme wild-type mrna 3 - UCGACG C ACU ACUCG AGCTGC A TGA GAGC-3 G G A C T CAAC G AG CT A Gefitini EGF p CL1-5 wt PC9 d T79M mrna 3 - UCGACGUACU ACUCG AGCTGCATGA GAGC-3 G G A C T CAAC G AG CT A Figure 1 Secondry structure of in complex with T79M mrna nd wild-type mrna. The ctlytic core of 1 23 (in lue) is flnked y two inding rms with sequences complimentry to T79M mrna (in lck). The sequences of wild-type mrna (upper pnel) nd T79M mrna (lower pnel), RY clevge site (rrow), nd muttion site (in red) re indicted., epiderml growth fctor receptor; mrna, messenger RNA. EGF p CL1-5 wt PC9 d from wild-type mrna nd specificlly downregulting T79M mrna, including DNAzyme which is cleved t higher rte ginst mrna with the point muttion A U positioned t the clevge junction thn one contining A C under simulted physiologicl conditions. 23, which exhiited the est performnce (unpulished dt), ws selected from these DNAzymes for susequent experiments. Arm I of contins 1-nucleotide complementry sequence spnning the muttion site of T79M mrna, with the clevge site locted four nucleotides wy from the muttion. A one-nucleotide mismtch of rm I with wild-type mrna wekens mrna degrdtion ctivity, llowing to discriminte the mutnt vrint from wild-type mrna. overcomes TKI resistnce in T79M lung cncer cells CL1-5 hrors wild-type (designted CL1-5 wt ). PC9 hs deletion in exon 19 (designted PC9 d19 ). H1975 contins T79M nd L858R muttions (designted ) nd T79M muttion in oth lleles ws confirmed y genomic DNA sequencing nlysis (Supplementry Figure S1). EGF-ctivted in CL1-5 wt cells or constitutively ctivted in PC9 d19 cells ws inctivted y tretment with Gefitini, potent inhiitor (Figure 2). However, the phosphoryltion level of in T79M mutnt cells ( ) ws not suppressed y Gefitini tretment. The result suggested tht ws more resistnt to Gefitini tretment. Trnsfection of H1975 TM/ cells with resulted in significnt repression of protein expression compred to tretment with control, control DNAzyme with no complementrity to ny known humn mrna (Figure 2). tretment cused no such repression in CL1-5 wt or PC9 d19 cells contining wild-type nd del19, respectively, demonstrting the llele-specific selectivity of the DNAzyme. The level of the ctivted form of (p) ws downregulted in prllel with tht of Figure 2 overcomes TKI resistnce in T79M mutnt cells. Gefitini (1 μmol/l; ) or - (1 nmol/l; ) treted cells were nlyzed y immunolotting with the indicted primry ntiodies listed on the left. EGF (1 ng/ml) ws dded to CL1-5 wt 15 min efore cell lysis., epiderml growth fctor receptor; TKI, tyrosine kinse inhiitors. totl. These results suggest tht cn circumvent the -TKI resistnce ttriutle to the T79M mutnt. selectively ttenutes T79M expression nd downstrem signling A549 hrors wild-type (designted A549 wt ). CL97 contins T79M nd G719A muttions (designted CL97 TM/GA ) nd T79M muttion in oth lleles ws confirmed y genomic DNA sequencing nlysis (Supplementry Figure S1). To exmine the llele selectivity of, we evluted mrna-knockdown efficiency in two cell lines hroring wild-type (A549 wt nd CL1-5 wt ) nd two cell lines hroring T79M ( nd CL97 TM/GA ). Totl RNA extrcted from cells trnsfected with ws sujected to quntittive reverse trnscription polymerse chin rection; trnsfections served s controls. As shown in Figure 3, tretment of nd CL97 TM/GA cells with significntly suppressed expression, decresing mrna levels to 32 nd 23%, respectively, of those in treted controls. In contrst, mrna expression ws only slightly inhiited in A549 wt (99% of control) nd CL1-5 wt (81% of control) cells. Thus, exhiited t lest 2.5- fold increse in knockdown efficiency towrd T79M mrna compred with its wild-type counterprt, demonstrting the high llele-discrimintion ility of this DNAzyme. Like other memers of the receptor tyrosine kinses fmily, inding to its extrcellulr lignds triggers receptor dimeriztion, tyrosine phosphoryltion of downstrem trget molecules, nd ctivtion of vrious signling pthwys, including signl trnsducer nd ctivtor of trnscription 3 Moleculr Therpy Nucleic Acids

3 Overcoming -TKI Resistnce with DNAzyme 3 Reltive mrna levels (%) p pstat3 STAT3 pakt AKT perk ERK A549 wt CL1-5 wt A549 wt CL1-5 wt CL97 TM/GA EGF Figure 3 Specific downregultion of T79M expression nd downstrem signling y. () RT-qPCR nlysis of mrna in cells treted with (n = 3). Cells were hrvested 48 hours fter trnsfection with or (1 nmol/l). The reltive mount of mrna ws normlized to ACTB mrna. The dt re presented s mens ± SD nd were nlyzed y Student s t-test. Asterisks denote sttisticl significnt differences (P.5). () Immunolot nlysis of nd its downstrem signling pthwys. Cells were hrvested 72 hours fter trnsfecting with 1 nmol/l or. in wild-type cells ws ctivted y dding 1 ng/ml EGF 15 minutes efore cell lystes were hrvested., epiderml growth fctor receptor; mrna, messenger RNA; RT-qPCR, quntittive reverse trnscription polymerse chin rection. (STAT3), AKT, extrcellulr signl-regulted kinse (ERK), nd others. 24 To exmine the inhiitory effects of on protein expression nd downstrem signling, we performed immunolot nlysis. Control did not ffect phosphorylted, totl, nd its downstrem sustrtes, including phosphorylted form of STAT3, AKT, nd ERK when compred to untreted group in ll four cell line exmined (Supplementry Figure S2). Thus, tretment P <.5* P <.5* CL97 TM/GA ws used s reference control for the following experiments. On the other hnd, inhiited protein expression in oth T79M mutnt cell lines ( nd CL97 TM/ GA ), with concurrent decrese in the phosphorylted form of (Figure 3, two pnels t the right). lso inhiited the downstrem ctivtion of STAT3, AKT, nd ERK without ffecting the totl mount of ech individul protein. After EGF tretment, remined its suppression effect on protein expression nd downstrem signling including, STAT3, nd ERK ut not AKT (Supplementry Figure S3). In contrst, protein levels in -treted groups did not differ from tht of -treted groups in A549 wt nd CL1-5 wt cells (Figure 3, two pnels t the left); the phosphorylted form of nd tht of its downstrem sustrtes were similrly unffected y tretment in A549 wt nd CL1-5 wt. induces lung cncer cell poptosis in n llelespecific mnner nd its downstrem signling pthwys regulte importnt cell functions, including cell prolifertion nd survivl. 3 To exmine the effects of on cell survivl, we counted cell numers fter trnsfection of or. In A549 wt nd CL1-5 wt cells, no differences in vile cell numer were seen etween - nd -trnsfected groups (Figure 4,). In contrst, the vile cell numer of T79M mutnt cells ( nd CL97 TM/GA ) ws significntly retrded y trnsfection (Figure 4c,d). To determine whether triggers poptosis in T79M mutnt cell lines, we immunolotted for poly ADP-riose polymerse (PARP) nd performed flow cytometry nlyses on nnexin V (AV)- nd propidium iodide (PI)-stined cells. The clevge of PARP is cused y incresed ctivity of cspse-3 nd serves s mrker for poptosis. 25 Immunolot nlyses showed tht the decrese in level induced y tretment ws ccompnied y concomitnt increse in cleved PARP in oth T79M mutnt cell lines ( nd CL97 TM/ GA ) compred with tht in -treted groups (Figure 4e). Dul stining with AV nd PI in conjunction with flow cytometry is commonly used method for evluting cell viility nd poptosis sttus. AV-positive cells re generlly regrded s poptotic cells, nd PI-positive cells s ded cells. A flow cytometric nlysis of nd CL97 TM/GA cells stined with AV nd PI showed 51 nd 53% AV-positive in -trnsfected groups; the corresponding vlues for -trnsfected cells were only 16 nd 22% (Figure 4f). A lrge proportion of PI-positive cells in the -trnsfected groups were lso AV positive (33 nd 25% for nd CL97 TM/GA cells, respectively), indicting tht the -induced cell deth ws the result of cellulr poptosis. Cholesterol modifiction enhnces the inhiitory effect of on cell viility Cholesterol modifiction of nti-micrornas (ntgomirs) hs een shown to increse their ility to escpe endosomes nd enhnce their likelihood of mking contct with their trgets in the cytosol. 26 Accordingly, we dded cholesterol-triethylene glycol (chol-teg) group on the 3 -end of DNAzymes (c nd c) nd then tested their ctivity in cell viility ssys. Modifiction of with chol-teg significntly

4 4 Overcoming -TKI Resistnce with DNAzyme Cell numers (1 5 cells) A549 wt Time (h) 72 Cell numers (1 4 cells) CL1-5 wt Time (h) 72 c Cell numers (1 4 cells) Time (h) P <.1* 72 d Cell numers (1 4 cells) CL97 TM/GA Time (h) P <.1* 72 e PARP cparp CL97 TM/GA f Cells (%) CL97 TM/GA AV + /PI + AV + /PI AV /PI + AV /PI Figure 4 induces poptosis in n llele-specific mnner. ( d) A549 wt (), CL1-5 wt (), (c), nd CL97 TM/GA (d) cell numers were determined t vrious times fter (1 nmol/l) trnsfection (n = 3). These dt re presented s men ± SD nd were nlyzed y Student s t-test. Asterisks denote sttisticl significnt differences (P.1). (e, f) induces poptosis in cells hroring T79M. nd CL97 TM/GA cells were nlyzed 72 hours fter trnsfection with or y immunolotting with the indicted ntiodies (e) or flow cytometry stined with AV nd PI (f). Counts re presented s percentges. AV, nnexin V; PI, propidium iodide. incresed the inhiitory effect on cell viility from 46 to 79% in cells with no loss of llele specificity (no inhiition in CL1-5 wt cells). Neither modified nor primitive inhiited cell viility (Figure 5). Immunolot showed tht the phosphorylted form of nd tht of its downstrem sustrtes were unffected y c tretment when compred to those in untreted group (Supplementry Figure S4). Thus, c tretment ws used s reference control for the following experiments. On the other hnd, c inhiited expression of T79M ut not wild-type (Figure 5), demonstrting tht the chol-teg-modified DNAzyme retined the ility to specificlly repress protein expression in n llele-specific mnner. Downstrem phosphoryltion of STAT3, AKT, nd ERK were lso inhiited. Moreover, the cleved form of PARP ws elevted in H1975 TM/ cells, indicting tht chol-teg modifiction did not lter the ility of to specificlly trigger poptosis in T79M mutnt cells. Besides, fter EGF tretment, c remined its suppression effect on protein expression nd downstrem signling including, STAT3, AKT, nd ERK (Supplementry Figure S5). Synergistic ntitumor effect of comined tretment with c nd BIBW-2992 in vitro nd in vivo c could e used s single gent or s prt of comintion therpy with second genertion TKIs to increse efficcy in the tretment of NSCLCs tht hror the T79M mutnt. 5 The efficcy of comined tretment with c nd the TKI, BIBW-2992, ginst T79M-sed drug resistnce ws evluted in vitro nd in vivo. To evlute the synergistic ntitumor effects of BIBW-2992 nd c, we used lower, suoptiml concentrtions of (5 nmol/l) nd BIBW-2992 (2 nmol/l) in these experiments. Immunolot ws performed to evlute protein expression nd trns-utophosphoryltion sttus of its downstrem signling proteins (Figure 6 nd Supplementry Figure S6). Both c nd BIBW-2992 lone inhiited growth nd signling in T79M mutnt cells ( nd CL97 TM/GA ), leit through different mechnisms. c specificlly degrded T79M mrna nd decresed protein levels, wheres BIBW-2992 inhiited utophosphoryltion of without ffecting protein level. At suoptiml concentrtions, individul drugs did not efficiently suppress downstrem signling. In contrst, comined tretment significntly suppressed the phosphoryltion of STAT3, AKT, nd ERK (Figure 6 nd Supplementry Figure S6). Furthermore, BIBW-2992 enhnced the cell-killing effect of c in concentrtion-dependent mnner on H1975 TM/ cells (5 nmol/l c in Figure 6 nd 25 nmol/l c in Supplementry Figure S7) nd CL97 TM/GA cells (25 nmol/l c in Supplementry Figure S7c). Comintion index (CI) vlues were clculted to evlute the comined effect of c nd BIBW This nlysis showed tht comined tretment exerted synergistic inhiitory effect (CI < 1) on the viility of cells (Figure 6c nd Supplementry Figure S7) nd CL97 TM/GA cells (Supplementry Figure S7d). Moleculr Therpy Nucleic Acids

5 Overcoming -TKI Resistnce with DNAzyme 5 Cell viility (%) ERK c c CL1-5 wt c c CL1-5 wt c Figure 5 Cholesterol modifiction of enhnces the inhiitory effect on cell viility of while retining its llele specificity. () CL1-5 wt nd cells were treted with 1 nmol/l with or without cholesterol modifiction. MTT ssys were performed 72 hours fter trnsfection to determine cell viility (n = 3). The dt re presented s men ± SD nd were nlyzed y Student s t-test. Asterisks denote sttisticl significnt differences (P.1). () Cell lystes were nlyzed y immunolotting. Synergistic effects of c nd BIBW-2992 were lso seen in the xenogrft niml model. Compred with the control group (c), ll three drug-treted groups (c+bibw-2992, c, nd c+bibw-2992) inhiited the growth of tumor originted from cells to different degrees (Figure 6d). Comined tretment with c nd BIBW-2992 showed the highest potency mong ll tretments in suppressing xenogrft tumor growth. In this group, the verge size of excised tumors ws pproximtely fourfold smller thn tht in the control group. An immunohistochemicl nlysis of tumor tissues showed severe necrosis in tumor tissues from the comined tretment group ut not in tissues c c c EGF + + p pstat3 STAT3 pakt AKT perk PARP cparp c P <.1* c from the control group (Figure 6e, upper pnel). in cells contins oth L858R nd T79M muttions, nd thus cn e detected using n ntiody specific for the L858R mutnt form. Tumor sections from the comined tretment group exhiited lower levels of L858R expression ccompnied y higher cspse-3 protein expression levels compred with sections from the control group (Figure 6e, middle nd lower pnel). Tumor tissues were lso evluted for expression nd downstrem signling. The results showed tht the comintion of c nd BIBW-2992 further suppressed totl expression, the phosphoryltion of in tumor tissues, nd the levels of the phosphorylted forms of STAT3, AKT, nd ERK compred with the control group (Figure 6f). The effect of c tretment on expression nd downstrem signling were evluted y immunolot regrding the tumor tissues from phosphte-uffered sline uffer group nd c-treted group. Immunolot showed tht the phosphorylted form of nd tht of its downstrem sustrtes were unffected y c tretment when compred to those in phosphte-uffered sline uffer group (Supplementry Figure S8). Tken together, these results indicte tht the comintion of c nd BIBW synergisticlly inhiits protein expression nd downstrem signling, triggering T79M-hroring cells to undergo poptosis nd suppressing xenogrft tumor growth. Discussion In this study, we demonstrted tht llele-specific is cple of overcoming -TKI resistnce ttriutle to secondry T79M muttion in NSCLC. We lso showed tht modifiction of with chol-teg enhnced its efficcy in inhiiting cncer cell growth. Furthermore, we showed synergistic effect of c nd BIBW-2992 on growth inhiition in vitro nd in vivo in cncer cells hroring the T79M mutnt. This study provides successful exmple of llele-specific gene silencing therpy overcoming T79M-sed drug resistnce. Since the first demonstrtion tht synthetic smll interfering RNAs (sirnas) induced n RNA interference effect in mmmlin cells y Thoms Tuschl, 27 there hs een surge in interest in hrnessing sirna for iomedicl reserch nd drug development. Limittions in sirna design hs, t times, impeded the genertion of sirnas tht re efficcious in degrding trget mrna, prticulrly with respect to llelespecific trgeting. In ddition, some mrnas re inherently reclcitrnt to perturtion y smll RNAs. 28 Given these limittions, DNAzymes provide super lterntive for gene silencing, prticulrly for pplictions tht require specific trgeting of mutnt site. Unlike sirna, which requires formtion of n RNA-induced silencing complex with Dicer protein to cleve mrna, divlent metl ions such s Mg 2+, which re undnt in the cell cytosol, re sufficient for DNAzymemedited ctlysis The dvntges of DNAzymes over sirnas re more resistnt to nuclese ttck, cheper to synthesize, nd esier to modify. 14 Modifictions, such s introduction of nonstndrd nucleotides 32 nd sustitution of linkge onds 22 or functionl groups, 14 cn e introduced into DNAzymes to enhnce trnsport efficiency, piring cpcity, or enzymtic ctivity.

6 6 Overcoming -TKI Resistnce with DNAzyme c c c c BIBW p pstat3 STAT3 pakt AKT perk ERK Cell viility (%) c c P =.7 P <.1* BIBW-2992 concentrtion (nmol/l) c CI Frction ffected d Tumor volume (mm 3 ) 1, c c+bibw-2992 c c+bibw Time (d) P <.5* * * * * f xenogrft e c c+bibw-2992 c c c c BIBW p H&E pstat3 STAT3 L858R pakt AKT perk Cspse-3 ERK Figure 6 Synergistic effects of c nd BIBW () Immunolot nlysis of cells treted with c or c (5 nmol/l) incuted with or without dded BIBW-2992 (2 nmol/l). (, c) MTT ssy of cells treted with c or c (5 nmol/l) comined with 25 ( ), 5 ( ), 1 ( ), or 25 nmol/l ( ) BIBW-2992 (n = 3). The dt re presented s the men ± SD. The results were nlyzed y Student s t-test nd CI clcultion. An sterisk denotes sttisticl significnt difference (P.1). (d f) Comined tretment silences signling, triggers poptosis, nd suppresses xenogrft tumor growth. (d) c (5 pmoles) ws intrtumorlly injected (twice per week) nd BIBW-2992 (2 mg/kg) ws orlly dministrted (three times per week) 1 dys (rrow indicted) fter inoculting xenogrft mice with (n = 7). The dt re presented s men ± SD nd were nlyzed y Student s t-test. Asterisks denote sttisticl significnt differences (P.5). (e) Xenogrft tumor tissues were processed for immunostining. Scle rs represent 2 μm in H&E imges nd 1 μm in L858R nd cspse-3 imges. (f) Xenogrft tumor tissues were processed for immunolotting. CI, comintion index;, epiderml growth fctor receptor. Moleculr Therpy Nucleic Acids

7 Overcoming -TKI Resistnce with DNAzyme 7 Owing to their highly negtively chrged nture, oligonucleotides re difficult to trnsfer cross cell memrnes. Cholesterol modifiction hs een demonstrted to enhnce the ility of ntgomirs to escpe endosomes nd increse the ccessiility of their trget RNA in the cytosol. 26 We evluted the efficcy of cholesterol-teg modifiction of in inhiiting cncer cell growth nd found tht this modifiction significntly enhnced the ntiprolifertive effect of without ffecting its llele specificity in distinguishing the T79M mutnt form from its wild-type counterprt. Cholesterol provides lipid moiety tht llows c to pss through cell memrnes more efficiently, wheres oth cholesterol nd TEG my enhnce resistnce to degrdtion y exonucleses in iologicl systems. We lso demonstrted tht c nd BIBW-2992 functioned in synergistic mnner ginst signling pthwys to suppress the growth of T79M-hroring NSCLC cells. The mechnisms of ction of c nd TKIs re different. c cleves T79M mrna, which leds to decrese in protein levels, wheres BIBW-2992 is second-line TKI tht irreversily inds to the intrcellulr kinse domin of nd suppresses its downstrem signling. A recent report showed tht support of cncer cell survivl is independent of its kinse ctivity, suggesting tht TKI lone could not chieve mximum therpeutic efficcy. 33 In ddition to its pivotl role in signling pthwys, lso intercts with nd stilizes the sodium/glucose cotrnsporter 1 to mintin intrcellulr glucose levels, which prevents ginst utophgic cell deth. c-induced inhiition of expression is dvntgeous compred with TKI effects in tht it my lso lock the kinse-independent functions of. Comintion therpies could llow the dosges of TKIs to e reduced nd reduce unwnted toxicity towrd norml cells. Thus, the llele-specific DNAzyme my solve the two min ostcles in trgeted therpy of NSCLC: dose-limiting toxicity nd T79M-sed drug resistnce. To investigte the mechnisms y which knockdown inhiits cncer growth, we used suoptiml concentrtions of c in this study (higher concentrtions of c completely erdicted cncer cells, leving no cells to study; unpulished dt). As demonstrted y Ci et l. 2 in series of preclinicl studies, DNAzymes re sfe nd well tolerted t dose s high s 1 μg for intrtumor injection, n mount more thn 2-fold greter thn we used in our mouse studies (4.5 μg). In this pper, we demonstrted tht is n llele-specific silencing gent ginst T79M mutnt mrna. In ddition to T79M, prevlent mutnts seen in NSCLC ptients (with popultion percentges in prenthesis) include G719S in exon 18 (5%), E746-A75 deletion in exon 19 (45%), nd L858R in exon 21 (4 45%). 4 Like, other DNAzymes cn e designed to knock down the expression of ech of the mutnt forms of in n llele-specific mnner without ffecting wild-type expression. muttions vry mong individul ptients nd cn chnge within n individul due to genetic ltertions tht occur during the development nd progression of cncer. Allele-specific DNAzymes could thus e used in comintion to knock down ll types of mrna vrints. They could lso e used in comintion with other drugs, such s -TKIs or -specific ntiodies, to completely erdicte cncer cells. Mterils nd methods Cell lines nd regents. A549 wt, CL1-5 wt,, CL97 TM/ GA, nd PC9 d19 cells were cultivted t 37 C with 5% CO 2 in RPMI-164 medium (Gico BRL; Life Technologies, Grnd Islnd, NY) supplemented with 1% (v/v) het-inctivted fetl ovine serum (Gico BRL). A549 wt,, nd PC9 d19 were purchsed from Americn Type Culture Collection (Mnsss, VA). CL1-5 wt is highly invsive humn lung denocrcinom derived from prentl CL Both CL1-5 wt nd CL97 TM/GA35 were developed in our lortory. Gefitini nd BIBW-2992 were kindly provided y Dr. Jmes Chih-Hsin Yng (Ntionl Tiwn University). MTT (3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrzolium romide) solution ws purchsed from (Sigm-Aldrich, St Louis, MO). Genomic DNA sequencing nlysis. Genomic DNA ws extrcted from nd CL97 TM/GA cell lines using QIA- GEN lood nd cell culture DNA kit ccording to the mnufcturer s protocol (Qigen, Hilden, Germny). The primer pirs used for PCR mplifiction of exon 2 were: 5 -CCA TGA GTA CGT ATT TTG AAA CTC-3 (forwrd) nd 5 -CCA CAC TGA GCA CTC AAT AAA GAG-3 (reverse). The purified PCR products were exmined y sequencing nlysis. DNAzyme synthesis. All DNAzymes were synthesized y Integrted DNA Technologies (Corlville, IA). The sequence of the control DNAzyme () showed no complementrity to ny known humn mrna. ws designed to hyridize specificlly to T79M mutnt mrna. The DNA sequences of nd re 5 -CAT CGG AGG CTA GCT ACA ACG AGA CAG CTG-3 nd 5 -AGC TGC ATG AGG CTA GCT ACA ACG AGA GC-3 (Figure 1). Phosphorothiote onds were introduced etween underlined nucleotides t oth ends of the DNAzymes. Chol-TEG ws covlently ttched to the 3 -end of nd (c nd c) t synthesis. Cell culture nd drug tretment. Cells were seeded onto sixwell pltes t cells per well. After culturing overnight, cells were treted with 1 nmol/l or mixed with Lipofectmine 2 (Invitrogen, Crlsd, CA). In immunolot experiments, for wild-type ctivtion nd inctivtion, A549 wt nd CL1-5 wt cells trnsfected with DNAzyme were serum-strved for 24 hours nd incuted with or without 1 ng/ml EGF t 37 C for 15 minutes immeditely efore cell lysis. To exmine the potentil EGF-medited signling in nd CL97 TM/GA cells, 1 ng/ml EGF ws dded 15 minutes efore cell lystes were hrvested. In TKI-resistnce tests, 1 μmol/l Gefitini ws dded into culture medium 6 hours efore cell collection. Only CL1-5 wt nd cells were used in chol-teg-modified DNAzyme studies. In order to leve enough surviving cells for nlysis, suoptiml concentrtion (5 nmol/l) of c or c ws used for trnsfections. In the comined tretment study, H1975 TM/ nd CL97 TM/GA cells were treted with 5 nmol/l c or

8 8 Overcoming -TKI Resistnce with DNAzyme c together with 2 nmol/l BIBW-2992 or dimethyl sulfoxide (vehicle control), dded to the culture medium. Quntittive reverse trnscription polymerse chin rection. Forty-eight hours fter trnsfection, totl RNA ws extrcted using TRIzol, ccording to the mnufcturer s protocol (Invitrogen). One-step reverse trnscription polymerse chin rection ws performed using the LightCycler 48 system (Roche Applied Science, Mnnheim, Germny). The reltive mount of mrna ws normlized to (ACTB) mrna. The sequences of PCR primer pirs were 5 -CAT CTC CGA AAG CCA ACA A-3 (forwrd) nd 5 -CTG CGT GAT GAG CTG CAC-3 (reverse) for, nd 5 -ATT GGC AAT GAG CGG TTC-3 (forwrd) nd 5 -GGA TGC CAC AGG ACT CCAT-3 (reverse) for ACTB. Immunolot. Totl protein ws extrcted 72 hours fter trnsfection using rdioimmunoprecipittion ssy uffer contining complete protese inhiitor cocktil (Roche Applied Science). For niml studies, xenogrft tumor tissues were lso lysed with rdioimmunoprecipittion ssy uffer. Protein concentrtions were determined using the Bio-Rd Protein Assy (Bio-Rd, Richmond, CA). Smples (5 μg protein) were resolved on 1% sodium dodecyl sulfte polycrylmide gels, trnsferred onto nitrocellulose memrnes, nd immunolotted with primry ntiodies. Primry ntiodies ginst the following proteins were used in immunolot nlyses t 1:1, dilution (the phosphoryltion site is given in prenthesis): (Snt Cruz Biotechnology, Snt Cruz, CA), p (Y168; Cell Signling Technology, Beverly, MA), STAT3 (Cell Signling Technology), pstat3 (Y75; Cell Signling Technology), AKT (Snt Cruz Biotechnology), pakt (S473; Cell Signling Technology), ERK (Snt Cruz Biotechnology), perk (T22/Y24; Cell Signling), nd cparp (cleved-parp [Asp214]; Cell Signling); nti- (Snt Cruz Biotechnology). Horserdish peroxidse conjugted nti-rit or mouse IgG (Snt Cruz Biotechnology) ws used s secondry ntiody t 1:5, dilution. Protein nds on memrnes were visulized using enhnced chemiluminescence regents (Amershm Phrmci, Pisctwy, NJ). Cell counting. Cells t exponentil growth phse were otined y seeding cells t different concentrtions nd cultivted in RPMI-164 medium supplemented with 1% (v/v) het-inctivted fetl ovine serum. Cells were trypsinized (Invitrogen) nd counted with hemocytometer, 24, 48, nd 72 hours fter trnsfection. Apoptosis ssy. Cells seeded onto 1-cm dishes were cultured overnight nd treted with 1 nmol/l or mixed with Lipofectmine 2. Cells were collected t 72 hours, doule stined with AV nd PI, nd nlyzed y flow cytometry (FACSCnto system; BD Biosciences, Sn Jose, CA) following the mnufcturer s protocol (Alex Fluor 488 Annexin V/Ded Cell Apoptosis Kit; Invitrogen). Cell viility ssy. Seventy-two hours fter or trnsfection, cells were rinsed three times with phosphte-uffered sline prior to incution with.5 mg/ml MTT solution t 37 C for 3 hours. MTT solution ws then replced with dimethyl sulfoxide, nd cell prolifertion ws determined y mesuring sornce t 57 nm with SpectrMx Plus384 Microplte Reder (Moleculr Devices, Sunnyvle, CA). Synergy. nd CL97 TM/GA cells were treted with 25, 5 nmol/l c or c together with 25, 5, 75, 1, 15, 25 nmol/l BIBW-2992 or dimethyl sulfoxide (vehicle control) dded to the culture medium. Seventy-two hours fter tretment, MTT ssys were performed to monitor cell viility. CI vlues were clculted y CI eqution of Chou Tlly using CompuSyn version 3..1 softwre (ComoSyn, Prmus, NJ). 36 CI= ( D ) 1 ( D) 2 + ( D ) ( D ) x 1 (D x ) 1 ws the dose of c lone tht inhiited x% of cell viility while (D x ) 2 ws the dose of BIBW-2992 lone tht inhiited x% of cell viility. (D) 1 ws the portion of c nd (D) 2 ws the portion of BIBW-2992 tht chieved x% of inhiition when comined tretment of c nd BIBW Frction ffected (F) on the x-xis of F-CI plot represented the frction of cell viility inhiition on drug treted cells. CI vlues greter thn 1, equl to 1, nd less thn 1 indicte ntgonistic effects, dditive effects, nd synergistic effects, respectively. 36 In vivo tumorigenesis ssy. All niml studies were performed ccording to protocols pproved y the Lortory Animl Center, Acdemi Sinic. Eight-week-old Bl/c nude mice (BioLASCO, Tipei, Tiwn) were sucutneously inoculted with cells (dy ). In the comined-tretment study, mice were rndomly divided into four groups on dy 1 nd dministered the following drug or drug comintions: (i) c, (ii) c+bibw-2992, (iii) c, or (iv) c+bibw Chol-TEG-modified DNAzyme (5 pmoles) mixed with Lipofectmine 2 ws injected intrtumorlly twice per week. BIBW-2992 ws suspended in phosphte-uffered sline nd dministered three times per week y orl gvge t 2 mg/kg. The length (L) nd width (W) of tumors were mesured with clipers every 3 4 dys, nd tumor volumes were clculted s (L W 2 )/2. After mice were scrificed, tumors were excised. Smll sections of tumors were processed for immunolot nd the remining tumor tissue ws fixed with 1% formlin nd emedded in prffin. Xenogrft tumor slides were stined with hemtoxylin nd eosin (H&E), nti- (L858R mutnt specific; Cell Signling), nd nti-cspse 3 (Cell Signling). Supplementry Mteril Figure S1. The muttion sttus of exon 2 in H1975 TM/ nd CL97 TM/GA cell lines. Figure S2. expression nd downstrem signling is unffected y tretment. Figure S3. remins its suppression effect on T79M expression nd downstrem signling fter EGF tretment in T79M mutnt cells. Figure S4. expression nd downstrem signling is unffected y c tretment in CL1-5 wt nd. x 2 Moleculr Therpy Nucleic Acids

9 Overcoming -TKI Resistnce with DNAzyme 9 Figure S5. c remins its suppression effect on T79M expression nd downstrem signling fter EGF tretment in. Figure S6. Comined tretment of c nd BIBW-2992 significntly suppresses the phosphoryltion of STAT3, AKT, nd ERK in CL97 TM/GA cells. Figure S7. Comined tretment of c nd BIBW-2992 exerts synergistic inhiitory effect on cell viility in cells hroring T79M mutnts. Figure S8. expression nd downstrem signling is unffected in xenogrft tissue fter c tretment. Acknowledgments. This work ws supported y grnts from the Ntionl Science Council of the Repulic of Chin (NSC B1-16, NSC B1-38, NSC I-2-33). The uthors hve declred tht no conflicts of interest exist. The uthors would like to dedicte this pper to the memory of Dr. Konn Peck, who pssed wy during the period of this reserch. Dr. Peck originted the rtionl design of DNAzymes; without his long-term devotion to lung cncer reserch, this pper could not hve een completed. 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