Targeting Hedgehog signaling pathway and autophagy overcomes ...

Targeting Hedgehog signaling pathway andautophagy overcomes drug resistance of BCR-

ABL-positive chronic myeloid leukemiaXian Zeng,1,2,y Hui Zhao,3,4,y Yubin Li,1,y Jiajun Fan,1 Yun Sun,1 Shaofei Wang,1 Ziyu Wang,1 Ping Song,1 and Dianwen Ju1,*

1Department of Biosynthesis and Key Laboratory of Smart Drug Delivery; MOE; School of Pharmacy; Fudan University; Shanghai, China; 2Bioinformatics and Drug Design Group;

Department of Pharmacy; Faculty of Science; National University of Singapore; Singapore; 3Department of Pharmacology; School of Pharmacy; Fudan University; Shanghai, China;4Department of Pharmacology; Yong Loo Lin School of Medicine; National University of Singapore; Singapore

yThese authors equally contributed to this work.

Keywords: autophagy, BCR-ABL, CML, drug resistance, hedgehog pathway

Abbreviations: ACTB, actin; b; AKT/protein kinase B, v-akt murine thymoma viral oncogene homolog; ATG, autophagy-related;Bafi A1, bafilomycin A1; BCC, basal cell carcinoma; BCR-ABL, breakpoint cluster region-ABL proto-oncogene, non-receptor tyro-sine kinase; CASP, caspase; apoptosis-related cysteine peptidase; CML, chronic myeloid leukemia; CQ, chloroquine; EIF4EBP1,

eukaryotic translation initiation factor 4E binding protein 1; HCQ, hydroxychloroquine; Hh, Hedgehog; MAP1LC3B, microtubule-associated protein 1 light chain 3 b; MTOR, mechanistic target of rapamycin; PARP, poly (ADP-ribose) polymerase; PBMC, humanperipheral blood mononuclear cell; PCR, polymerase chain reaction; RPS6KB, ribosomal protein S6 kinase, 70kDa; siRNA, small

interfering RNA; SQSTM1, sequestosome 1; TKI, tyrosine kinase inhibitor.

The frontline tyrosine kinase inhibitor (TKI) imatinib has revolutionized the treatment of patients with chronicmyeloid leukemia (CML). However, drug resistance is the major clinical challenge in the treatment of CML. TheHedgehog (Hh) signaling pathway and autophagy are both related to tumorigenesis, cancer therapy, and drugresistance. This study was conducted to explore whether the Hh pathway could regulate autophagy in CML cells andwhether simultaneously regulating the Hh pathway and autophagy could induce cell death of drug-sensitive or-resistant BCR-ABLC CML cells. Our results indicated that pharmacological or genetic inhibition of Hh pathway couldmarkedly induce autophagy in BCR-ABLC CML cells. Autophagic inhibitors or ATG5 and ATG7 silencing couldsignificantly enhance CML cell death induced by Hh pathway suppression. Based on the above findings, our studydemonstrated that simultaneously inhibiting the Hh pathway and autophagy could markedly reduce cell viability andinduce apoptosis of imatinib-sensitive or -resistant BCR-ABLC cells. Moreover, this combination had little cytotoxicity inhuman peripheral blood mononuclear cells (PBMCs). Furthermore, this combined strategy was related to PARPcleavage, CASP3 and CASP9 cleavage, and inhibition of the BCR-ABL oncoprotein. In conclusion, this study indicatedthat simultaneously inhibiting the Hh pathway and autophagy could potently kill imatinib-sensitive or -resistant BCR-ABLC cells, providing a novel concept that simultaneously inhibiting the Hh pathway and autophagy might be a potentnew strategy to overcome CML drug resistance.

Introduction

Chronic myeloid leukemia (CML) is a clonal disease which isassociated with aberrant expression of the chimeric protein BCR-ABL.1 Although the frontline TKI imatinib revolutionized thetreatment of patients with CML, acquired imatinib resistancemainly resulting from BCR-ABL gene mutation is an emergingproblem,2,3 and remains to be resolved. New TKIs dasatinib andnilotinib overcame this problem to some extent but had no effecton the drug-resistant T315I mutation in CML patients. Theinvestigation of new regimes or combinational therapies

improving the current condition of CML treatment would pro-vide more options for patients and benefit the clinical cure ofCML.

The Hedgehog (Hh) pathway, which can be categorized into3 subgroups: Desert Hedgehog (Dhh), Indian Hedgehog (Ihh), andSonic Hedgehog (Shh), plays crucial roles in growth, survival, andfate of vertebrate cells.4,5 In mammalian cells, Hh, PTCH(patched), SMO (smoothened, frizzled class receptor), and GLI/glioma-associated oncogene (GLI family zinc finger) transcrip-tion factors (GLI1, GLI2, and GLI3) are key components of theHh pathway.6 Primary cilium provides the crucial location where

*Correspondence to: Dianwen Ju; Email: [email protected]: 02/12/2014; Revised: 07/24/2014; Accepted: 11/18/2014http://dx.doi.org/10.4161/15548627.2014.994368

www.tandfonline.com 355Autophagy

Autophagy 11:2, 355--372; February 2015; © 2015 Taylor & Francis Group, LLCTRANSLATIONAL RESEARCH PAPER

Hh signaling is regulated. PTCH localizes to the primary ciliumand inhibits SMO activity by preventing its trafficking and locali-zation to the primary cilium, resulting in proteasomal cleavage ofGLI and consequently leading to the inhibition of Hh signalingpathway.7 The Hh ligand can bind to PTCH, displace PTCHfrom the primary cilium, and trigger the activation of SMO;acti-vated SMO can subsequently lead to the activation of the Hh sig-naling pathway.7

Increasing evidence indicates that aberrant activation of Hhpathway is closely linked to tumorigenesis.6 It is well establishedthat abnormal activation of the Hh pathway is responsible formultiple cancers including basal cell carcinoma (BCC), hemato-logic malignancies, breast cancer, gastric cancer, and pancreaticcancer.4,6-9 Therefore, the Hh signaling pathway has been one ofthe most promising anti-cancer targets, and several Hh inhibitorsare being evaluated in ongoing clinical trials for many types ofcancers.10 Vismodegib (GDC-0449, a systemic antagonist ofSMO, Roche) is the first small-molecule, Hh-pathway inhibitor,which has been approved by the US. Food and Drug Administra-tion for the treatment of BCC. There are close relationshipsbetween the CML and the Hh pathways.11-15 Evidence showsthat the abnormal activation of the Hh pathway is critical for theexpansion of BCR-ABLC leukemia stem cells.16 The Hh pathwayinhibitor cyclopamine can induce cell death of leukemia cells invitro.17 Inhibition of Hh pathway could reduce drug resistanceof CD34C leukemia cells in vitro.18 Therefore, the combinationof Hh pathway inhibitors and TKIs might represent a hopefulstrategy for improving treatment outcomes of patients withCML. The combination of PF-04449913 (an Hh inhibitor,Pfizer) with Dasatinib for CML treatment is under phase I clini-cal trial (ClinicalTrials.gov: NCT00953758). A recent studyreports that the combination of ponatinib and the Hh inhibitorvismodegib can induce cell death in drug-resistant CML cells.19

However, this combined therapy might be based on the efficacyof TKIs themselves, since ponatinib used alone could kill drug-resistant CML cells. New strategies are needed to make any prog-ress in the field of CML drug resistance.

Macroautophagy (autophagy hereafter) is a highly controlledbulk proteolytic process that can be induced by many conditionsof stress such as nutrient or growth factor deprivation, oxidativestress, and drug treatment, and is being increasingly investigatedas an anticancer target.20-22 Increasing evidence indicates thatautophagy plays a critical role in tumorigenesis, progression, andtreatment of hematological malignancies.23,24 Inhibition ofautophagy enhances imatinib’s cytotoxicity on Philadelphia chro-mosome-positive cells. However, inhibiting autophagy cannotincrease the sensitivity of BCR-ABLT315I-harboring leukemiacells to TKIs.24,25 Moreover, a clinical trial is ongoing to evaluatethe efficacy of combination therapy of imatinib with HCQ onCML (ClinicalTrials.gov Identifier: NCT01227135, phase II).26

Of note, recent reports have reported Hh pathway can regulateautophagy, while the conclusions were controversial: Activationof the Hh signaling pathway using Hh agonists can inhibitautophagy in HeLa cells, and inactivation of Hh-inducedautophagy in Drosophila.27 Inhibition of the Hh pathway usingGANT61, an Hh pathway inhibitor which is undergoing phase

II clinical trial, can induce autophagy in human hepatocellularcarcinoma cells.28 This evidence indicates that the Hh pathwayhas an inhibitory effect on autophagy. However, the Hh pathwayexhibits a positive regulatory effect on autophagy in vascularsmooth muscle cells and hippocampal neurons.29,30 These find-ings suggest that there might be extensive interactions betweenthe Hh pathway and autophagy in cancers including hematologi-cal malignancies and that these interactions might be utilized forimproving cancer therapy. Therefore, we hypothesized thatsimultaneously targeting the Hh pathway and autophagy mightrepresent a novel turning point in cancer therapy.

In our current study, we investigated the relationship betweenthe Hh pathway and autophagy in CML and the potential ofovercoming CML drug resistance based on combined targetingof Hh pathway and autophagy. Our results showed that inhibit-ing the Hh pathway and autophagy could remarkably induce celldeath of drug-sensitive and -resistant BCR-ABLC CML cells.First, we found that inhibiting the Hh pathway could induceautophagy in BCR-ABLC CML cells. Second, compared withinhibition of the Hh pathway or autophagy alone, inhibiting theHh pathway and autophagy simultaneously could sharplydecrease the cell viability of BCR-ABLC cells and significantlyinduce apoptosis. Moreover, our results suggested that inhibitionof the Hh pathway and autophagy downregulated the kinaseactivity of the BCR-ABL oncoprotein. This study provided anovel notion that beyond TKIs, simultaneously inhibiting theHh pathway and autophagy might be a potential new strategy toovercome CML drug resistance.

Results

Inhibition of the Hh pathway reduced cell viability of BCR-ABLC CML cells

Initially, we determined whether the Hh inhibitor vismodegibcould effectively inhibit the Hh pathway in CML cells. It is diffi-cult to detect the change of PTCH expression in the protein leveldue to the lack of a specific antibody against endogenous PTCH.Therefore, we detected the relative expression of mRNA levels ofPTCH. Quantitative RT-PCR results showed that 10, 20, and40 mM of vismodegib could significantly minimize the relativeexpression of PTCH and GLI1 mRNA, indicating that the Hhpathway was inhibited by vismodegib (Fig. 1 A and B). It is wellaccepted that the expression level of GLI1 can reflect the activa-tion status of the entire Hh pathway.6 Our results showed thatthe Hh inhibitor vismodegib could appreciably decrease the pro-tein level of GLI1 at the concentrations of 10, 20, and 40 mM,suggesting the inhibition of Hh pathway in CML cells (Fig. 1C).

Although the comprehensive elucidation of the upstream anddownstream of Hh signaling is insufficient, present evidenceindicates that, in CML, the Hh pathway upregulated the canoni-cal WNT signaling, CCND1 and MYC.4,7,31 Therefore, weexamined whether these protein targets were also affected by vis-modegib in CML cells. Western blot results showed that the pro-tein levels of CCND1 and MYC were decreased by vismodegibin a dose-dependent manner (Fig. 1C). In conclusion,

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vismodegib effectively inhibited the Hhpathway and its downstream protein tar-gets in CML cells.

Similarly to the Hh pathway, theWNT pathway is also one of the mostimportant signaling pathways that playskey roles in embryonic development,and is required for the cancer stem cells(CML stem cells) and CML progres-sion.32-35 The Hh pathway can interactwith the WNT pathway through phos-phorylating GSK3B.31 Western blotassays indicated that vismodegib aug-mented the phosphorylation of GSK3Band reduced the protein level ofCTNNB1, the key mediator of WNTsignaling, indicating the inhibition ofthe WNT pathway (Fig. 1C).

We also examined the inhibitoryeffects of vismodegib on cell viability indrug-sensitive and -resistant CML cells.The T315I and Y253F mutations ofBCR-ABL are 2 representative imatinib-resistant genotypes, while wild-typeBCR-ABL is an imatinib-sensitive geno-type. BaF3-BCR-ABL, BaF3-BCR-ABLT315I and BaF3-BCR-ABL YY253F

cells derived from BaF3 cells (a mousepro-B cell line) transfected with theBCR-ABL wild-type gene, the BCR-ABLT315I mutation gene and the BCR-ABLY253F mutation gene, respectively,are widely used in CML investiga-tions.36-38 Inhibiting the Hh pathwayusing the Hh inhibitor vismodegibcould reduce the cell viability of K562,BaF3-BCR-ABL, BaF3-BCR-ABLT315I,and BaF3-BCR-ABLY253F cells in vitroat the concentration of 40 mM (Fig. 1D).

Collectively, these data demonstrated that inhibition of theHh pathway could reduce the cell viability of BCR-ABLC CMLcells.

Inhibiting the Hh pathway induced autophagy in BCR-ABLC CML cells

Next, we tested whether autophagy could be induced byinhibiting the Hh pathway in BCR-ABLC CML cells. Usingtransmission electron microscopy, a gold standard of autophagyassessment, we observed more autophagosomes characterized bydouble membranes in BCR-ABLC CML cells exposed to vismo-degib when compared with controls (Fig. 2A), suggesting thatvismodegib induced autophagy in BCR-ABLC cells. To confirmvismodegib-induced autophagy, BCR-ABLC CML cells weretransiently transfected with an enhanced green fluorescent pro-tein (eGFP)-MAP1LC3B plasmid, which is a well-documentedmodel for monitoring the conversion of MAP1LC3B from a

cytosolic MAP1LC3B-I form to a phagophore- and autophago-some-bound MAP1LC3B-II form.39 Under normal conditions,eGFP-MAP1LC3B fluorescence is diffuse in the cytoplasm, whileit converts to a punctate pattern when autophagy is activated.Treatment with 20 mM of vismodegib for 24 h appreciablyincreased the average number of fluorescent spots (Fig. 2B), indi-cating the conversion of MAP1LC3B-I to MAP1LC3B-II.

The increase of autophagosomes or MAP1LC3B-II does notrepresent the completion of the entire autophagy pathway. Tofurther investigate if autophagy was induced after vismodegibtreatment, we examined the autophagic flux in vismodegib-treated BCR-ABLC CML cells. SQSTM1 is an extensively usedautophagy marker. Western blot assays showed the decrease ofSQSTM1 and increase of MAP1LC3B-II protein levels in vismo-degib-treated CML cells at several time points (Fig. 3A and B).We also observed the increase of MAP1LC3B-II in BCR-ABLC

CML cells in a dose-dependent manner (Fig. S1). Moreover, we

Figure 1. Inhibiting the Hh pathway decreased cell viability of BCR-ABLC CML cells. (A and B) K562cells were treated with 10, 20, and 40 mM of vismodegib for 24 h, gene expression of PTCH (A) andGLI1 (B) were detected by quantitative RT-PCR. Data shown are mean § SD of triplicates of one typi-cal experiment. Similar results were obtained from 3 independent experiments * versus Control, P <

0.05, ** vs. to Control, P < 0.01. (C) K562 cells were treated with 10, 20, and 40 mM of vismodegib for48 h, protein levels of GLI1, CCND1, MYC, p-GSK3B, GSK3B, CTNNB1, and ACTB were determined bywestern blot assay. Densitometric values were quantified using the ImageJ software and normalizedto control. The values of control were set to 1. The data represents the mean of 3 independent experi-ments. (D) K562, BaF3-BCR-ABL, BaF3-BCR-ABLT315I, and BaF3-BCR-ABLY253F cells were treated with 2.5,5, 10, 20, and 40 mM of vismodegib for 48 h, cell viability was determined by the CCK-8 assay.


used bafilomycin A1 (Bafi A1), a lysosomotropic agent, to pre-vent the fusion of autophagosomes with lysosomes. Treatmentwith vismodegib increased the protein levels of MAP1LC3B-II

and decreased the proteinlevels of SQSTM1 in CMLcells (Fig. 3C). When cellswere treated with vismode-gib in combination withBafi A1, the vismodegib-induced increase ofMAP1LC3B-II was signifi-cantly enhanced and the vis-modegib-induced decreaseof SQSTM1 was recoveredin CML cells (Fig. 3C).These data indicated thatvismodegib induced auto-phagic flux, which couldtrigger lysosomal degrada-tion of MAP1LC3B-II.

Moreover, small interfer-ing RNA (siRNA) assayswere conducted to furtherunderstand the role of inhib-iting the Hh pathway in vis-modegib-induced autoph-agy. Vismodegib inhibits theHh pathway through bind-ing to SMO. Therefore, wesilenced SMO to inhibit theHh pathway in CML cells.Owing to the lack of a spe-cific antibody against endog-enous SMO, to determinethe efficiency of SMO silenc-ing, the relative mRNA levelof SMO was measured byquantitative RT-PCR andthe protein levels of GLI1,CCND1, and MYC weredetermined by western blot.The results showed that therelative mRNA levels ofSMO (Fig. 4A) and theprotein levels of GLI1,CCND1, and MYC(Fig. 4B) were significantlyattenuated in cells trans-fected with SMO siRNA,compared with cells trans-fected with the nonsilencingscrambled control (SCR)siRNA, indicating that SMOsiRNA effectively silencedSMO and inhibited the Hhpathway. Consistent with

vismodegib treatment, inhibiting the Hh pathway using SMOsiRNA could also reduce the cell viability of CML cells(Fig. 4C). Moreover, we explored whether Hh pathway

Figure 2. Inhibiting the Hh pathway induced autophagy in CML cells. (A) K562, BaF3-BCR-ABL, BaF3-BCR-ABLT315I,and BaF3-BCR-ABLY253F cells were treated with 20 mM of vismodegib for 48 h, autophagosomes were observedusing transmission electron microscopy. Arrows represent autophagosomes. (B and C) K562, BaF3-BCR-ABL, andBaF3-BCR-ABLT315I cells were transiently transfected with an eGFPMAP1LC3B plasmid for 24 h, followed by treat-ment of 20 mM of vismodegib for 48 h. Cells were observed under confocal microscopy and typical images are pre-sented (B). Green fluorescence spots represent MAP1LC3B-II spots. MAP1LC3B-II spots were counted using ImageJsoftware (C). ** P< 0.01. n D 20.


inhibition by SMO silencing couldinduce autophagy in CML cells.Compared with cells transfected withSCR siRNA, the protein level ofMAP1LC3B-II increased when cellswere transfected with SMO siRNA(Fig. 4B). The results showed thatcompared with cells transfected withSCR siRNA, cells transfected withSMO siRNA showed more eGFP-MAP1LC3B fluorescence spots(Fig. 4D) and a higher protein levelof MAP1LC3B-II, and CQ could sig-nificantly further increase the proteinlevel of MAP1LC3B-II (Fig. 4E).These data confirmed that Hh path-way suppression could induce autoph-agy in CML cells. Taken together,these results demonstrated that inhib-iting the Hh pathway inducedautophagy in BCR-ABLC CML cells.

Downregulation of the AKT-MTOR signaling pathway wasinvolved in autophagy induced byinhibition of the Hh signaling pathway

The AKT-MTOR signaling pathway is a key autophagy-regu-lating pathway in eukaryotic cells.40 MTORC1 (MTOR com-plex 1) is involved in both the Hh pathway and autophagy.MTORC1 is a negative regulator of autophagy and its activationstatus can be evaluated by measuring the phosphorylation of itsdownstream targets including EIF4EBP1 (eukaryotic translationinitiation factor 4E binding protein 1) and RPS6KB (ribosomal

S6 protein kinase, 70kDa).41 Therefore, we investigated whethervismodegib affected the activation status of the MTOR signalingpathway. Vismodegib significantly decreased the protein levels ofphosphorylated AKT, MTOR, RPS6KB, and EIF4EBP1 in adose-dependent manner (Fig. 5A and B). Meanwhile, treatmentwith vismodegib for 24 h and 48 h appreciably decreased theprotein levels of phosphorylated AKT, MTOR, EIF4EBP1, andRPS6KB when compared with control groups (Fig. 5C and D),

Figure 3. Inhibiting the Hh pathwayinduced autophagic flux in CML cells. (A)K562 and BaF3-BCR-ABL cells weretreated with 20 mM of vismodegib for6 h, 12 h, 24 h, or 48 h, protein levels ofSQSTM1, MAP1LC3B, and ACTB weremeasured by western blot assays.(B) BaF3-BCR-ABLT315I and BaF3-BCR-ABLY253F cells were treated with 20 mMof vismodegib for 6 h, 12 h, 24 h, or48 h, protein levels of SQSTM1,MAP1LC3B, and ACTB were measured bywestern blot assays. (C) K562, BaF3-BCR-ABL, BaF3-BCR-ABLT315I, and BaF3-BCR-ABLY253F cells were cotreated with vis-modegib (20 mM) and Bafi A1 (5 nM) ortreated with these 2 agents alone for48 h, protein levels of SQSTM1,MAP1LC3B, and ACTB were measured bywestern blot assays. (A–C) Densitometricvalues were quantified using the ImageJsoftware and normalized to control. Thevalues of control were set to 1. The dataare presented as means § SD of 3 inde-pendent experiments.


Figure 4. For figure legend, see page 361.


indicating the inhibition of the AKT-MTOR signaling pathwayin CML cells.

Pharmacologically blocking autophagy could significantlyenhance the decrease in viability induced by the Hh inhibitor inBCR-ABLC CML cells

We used CQ to inhibit vismodegib-induced autophagy, sinceCQ inhibits autophagy through blocking the fusion of autopha-gosome and lysosome, which leads to the block of degradation ofMAP1LC3B-II and results in the increase of MAP1LC3B-II pro-tein level.39 Western blot results showed that the protein level ofMAP1LC3B-II in vismodegib- and CQ-cotreated cells washigher than cells treated with vismodegib alone (Fig. 6B), indi-cating that CQ inhibited vismodegib-induced autophagy inBCR-ABLC cells. Vismodegib combined with CQ significantlydecreased cell viability when compared with either agent alone(Fig. 6A and C). Moreover, the combination of vismodegib andBafi A1, another autophagy inhibitor, could also significantlydecrease cell viability of BCR-ABLC cells (Fig. S2). Furthermore,CQ could markedly enhance the inhibition of cell viabilityinduced by SMO-silencing (Fig. 4F). The Hh pathway is closelyrelated to CML cell growth. Thus, we studied whether the com-bination of vismodegib and CQ influenced cell cycle distributionin CML cells. Results showed that the percentage of G0/G1 phasecells did not change significantly between cells treated with bothvismodegib and CQ and cells treated with vismodegib alone(Fig. S3). Taken together, these results indicated that blockingautophagy could significantly enhance the inhibition of cell via-bility induced by the Hh inhibitor in BCR-ABLC CML cells.

ATG5 and ATG7 silencing significantly enhanced theinhibition of cell viability induced by the Hh inhibitor in BCR-ABLC CML cells

To further corroborate the crucial role of autophagy in theinduction of cell death induced by Hh inhibition, we used siRNAtargeting 2 established core autophagy molecules, namely ATG5and ATG7, in K562 (imatinib-sensitive) and BaF3-BCR-ABLT315I (imatinib-resistant) cells, respectively. As shown inFigure 7A and D, compared with transfection with the SCRsiRNA, a robust downregulation of ATG5 and ATG7 proteinlevels was observed 24 h after ATG5 or ATG7 siRNA transfec-tion. Meanwhile, Hh inhibiting-mediated autophagosome for-mation was significantly abrogated following ATG5 and ATG7silencing as evaluated by measuring MAP1LC3B conversion

(Fig. 7B and E). Next, we examined the cell viability under simi-lar experiment conditions. Although, in the absence of vismode-gib, there were significant inhibitions of cell viability after ATG5and ATG7 silencing when compared with SCR-transfected cells,robust cell viability inhibition was obtained in the presence of vis-modegib (Fig. 7C and F), demonstrating the critical role ofautophagy in the induction of CML cell death induced by Hhinhibition.

The simultaneous inhibition of the Hh pathway andautophagy could significantly inhibit cell viability of drug-resistant BCR-ABLC CML cells

We next focused our study on evaluating the effect of simulta-neously inhibiting the Hh pathway and autophagy on drug-resis-tant cells. Currently, drug resistance resulting from genemutations of BCR-ABL is a drawback of CML treatment. Amonga variety of mutations, the T315I mutation of BCR-ABL is resis-tant to most TKIs. Therefore, BaF3-BCR-ABLT315I cells wereused to evaluate the efficacy of overcoming CML drug resistancethrough the simultaneous inhibition of the Hh pathway andautophagy. The results showed that the combination of vismode-gib with CQ could significantly inhibit cell viability of drug-sen-sitive (K562) and -resistant BCR-ABLC (BaF3-BCR-ABLT315I)cells, whereas imatinib alone had no effect on drug-resistantBCR-ABLC cells (Fig. 8A and B). Consistent with the results ofthe previous study, our data indicated that the combination ofimatinib with CQ could significantly inhibit cell viability ofdrug-sensitive BCR-ABLC cells when compared with the treat-ment of imatinib or CQ alone, except for drug -resistant BCR-ABLC cells. Moreover, the combination of vismodegib with ima-tinib could also significantly inhibit cell viability of drug-sensitiveBCR-ABLC cells, except for drug-resistant BCR-ABLC cells.Importantly, the combination of vismodegib and CQ could sig-nificantly inhibit cell viability of drug-sensitive and -resistantBCR-ABLC cells (Fig. 8C). Furthermore, although vismodegibcould inhibit cell viability of PBMCs, there was no significantdifference between the group of cells treated with vismodegibalone and the group of cells cotreated with vismodegib and CQ(Fig. 8D). Since vismodegib has proven to be a safe drug in clini-cal use, this result suggested that this combined therapy might bea relative safe treatment strategy. Collectively, these data indi-cated that simultaneously inhibiting the Hh pathway andautophagy could effectively inhibit cell viability of drug-resistantBCR-ABLC CML cells.

Figure 4 (See previous page). SMO silencing induced autophagy in CML cells. (A to C) K562 cells were transiently transfected with SMO siRNA for 48 h.(A) The relative expression level of SMO mRNA was determined by quantitative RT-PCR. Data represents mean § SD of 3 independent experiments. **versus siSCR, P < 0.01. (B) The protein levels of GLI1, CCND1, MYC, MAP1LC3B, and ACTB were detected by western blot. Densitometric values werequantified using the ImageJ software and normalized to control. The values of control were set to 1. The data are presented as means of 2 independentexperiments. (C) Cell viability was measured by the CCK-8 assay. Data are presented as the means § SD of 3 independent experiments. ** vs. siSCR, P <

0.01. (D) After K562 cells were transiently transfected with an eGFPMAP1LC3B plasmid for 24 h, cells were transfected with SMO siRNA for 48 h.MAP1LC3B-II green fluorescent spots were observed under confocal microscopy and typical images are presented. ** P < 0.01. n D 20. (E and F) K562cells were transiently transfected with SMO siRNA for 24 h and followed by the treatment of CQ (10 mM) for 48 h. (E) The protein levels of SQSTM1,MAP1LC3B, and ACTB were measured by western blot assay. Densitometric values were quantified using the ImageJ software and normalized to control.The values of control were set to 1. The data are presented as means of 2 independent experiments. (F) Cell viability was measured by CCK-8 assay. Dataare presented as the means § SD of 3 independent experiments. **P < 0.01.


Simultaneous inhibition of the Hh pathway and autophagysharply induced apoptosis of BCR-ABLC CML cells

The Hedgehog pathway inhibitor can induce apoptosisin K562 cells.17 We investigated if simultaneously inhibitingthe Hh pathway and autophagy could induce apoptosis of

BCR-ABLC CML cells. The effect of the combination of vismo-degib with CQ on BCR-ABLC CML cells was determined bywestern blot and flow cytometry. Western blot assays showedthat the combination of vismodegib with CQ noticeably inducedthe cleavage of CASP9, CASP3, and its substrate PARP1 (poly

Figure 5. Vismodegib inhibited the AKT-MTOR signaling pathway in CML cells. (A) K562 cells were treated with 10, 20, and 40 mM of vismodegib (Vis) for48 h, the protein levels of p-AKT (Ser473), p-AKT (Thr308), p-MTOR (Ser2448), p-RPS6KB1 (Thr389), p-EIF4EBP1 (Thr37/46), SQSTM1, MAP1LC3B, and ACTBwere measured by western blot assays. (B) Densitometric values were quantified using the ImageJ software and normalized to control. The values of con-trol were set to 1. The data are presented as means § SD of 3 independent experiments. (C) K562 cells were treated with or without 20 mM of vismode-gib for 12 h, 24 h, and 48 h, the protein levels of p-AKT (Ser473), p-AKT (Thr308), p-MTOR (Ser2448), p-RPS6KB1 (Thr389), p-EIF4EBP1 (Thr37/46),SQSTM1, MAP1LC3B, and ACTB were measured by western blot assays. (D) Densitometric values were quantified as described in (B).


Figure 6. For figure legend, see page 364.


[ADP-ribose] polymerase 1) (Fig. 9A), suggesting that this com-bination activated the apoptosis pathway. Flow cytometry analy-sis showed that the proportions of apoptotic cells (ANXA5/annexin VC PI¡) and necrotic cells (ANXA5C PtdInsC) increasedsignificantly when BCR-ABLC cells were cotreated with vismode-gib and CQ for 24 h and 48 h (Fig. 9B). Moreover, the collapseof mitochondrial membrane potential, an indicator of early apo-ptosis, was detected after K562 cells were treated with vismode-gib and CQ (Fig. S4). These results suggested that combinationof vismodegib with CQ markedly induced apoptosis in BCR-ABLC cells. To confirm this observation, z-VAD-fmk, a pan-CASP inhibitor, was used to inhibit the cleavage of CASP. Imati-nib used as positive and negative controls of CASP activation in

K562 (imatinib sensitive) and BaF3-BCR-ABLT315I (imatinibresistant) cells, respectively. Western blot results indicated thatthe cleavage of CASP3 was abolished by 20 mM of z-VAD-fmk(Fig. 10A). However, 20 mM of z-VAD-fmk could not rescueCML cells from the reduction of cell viability induced by combi-nation of vismodegib and CQ (Fig. 10B), while z-VAD-fmkcould partially but significantly weaken the apoptosis induced bycombination of vismodegib and CQ (Fig. 10C). These data indi-cated that the CML cell death induced by combination of vismo-degib and CQ was partially depended on CASP-dependentapoptosis.

Furthermore, HL-60 cells, a BCR-ABL negative leukemia cellline, were used as independent control cells. Another group has

Figure 7. ATG5 and ATG7 silencing significantly enhanced cell death induced by Hh inhibition in CML cells. (A) K562 cells were transiently transfectedwith ATG5 or ATG7 siRNAs for 48 h, the protein levels of ATG5, ATG7, and ACTB were detected by western blot assays. (B) After K562 cells were transientlytransfected with ATG5 or ATG7 siRNAs for 24 h, cells were treated with 20 mM of vismodegib for 12 h, 24 h, and 48 h, the protein levels of SQSTM1,MAP1LC3B, and ACTB were measured by western blot assay. (C) K562 cells were treated as described in (B), cell viability was determined by CCK-8 assay.(D) BaF3-BCR-ABLT315I cells were treated as described in (A), the indicated protein levels were detected by western blot assay. (E) BaF3-BCR-ABLT315I cellswere treated as described in (B), the indicated protein levels were detected by western blot (E) and cell viability was measured by CCK-8 assay (F). Rela-tive expression levels in (A, B, D and E), shown below the blot are representative of 2 independent experiments. Densitometric values were quantifiedusing the ImageJ software and normalized to control. The values of control were set to 1. Data in (C and F) are means § SD of 3 independent experi-ments performed in triplicate. **P< 0.01.

Figure 6 (See previous page). The decrease in cell viability induced by the Hh inhibitor was significantly enhanced by inhibiting autophagy. (A) K562,BaF3-BCR-ABL, BaF3-BCR-ABLT315I, and BaF3-BCR-ABLY253F cells were treated with 20 mM of vismodegib and 2.5, 5, or 10 mM of CQ for 48 h, cell viabilitywas measured by CCK-8 assay. Data are means § SD of triplicates. Typical results of 3 independent experiments are presented. *P < 0.05; **P < 0.01. (Band C) K562, BaF3-BCR-ABL, BaF3-BCR-ABLT315I, and BaF3-BCR-ABLY253F cells were cotreated with vismodegib (20 mM) and CQ (10 mM) or treated withvismodegib (20 mM) or CQ (10 mM) alone for 48 h, (B) then the protein levels of MAP1LC3B, and tubulin were measured by western blot assays, (C) cellswere observed under inverted phase contrast microscope. Phase-contrast pictures were obtained randomly and representative pictures are presented.


reported that the Hh inhibitor cyclopamine can induce cell deathin HL-60 cells.42 We observed that vismodegib upregulated theexpression level of MAP1LC3B-II in HL-60 cells (Fig. S5A),indicating that inhibiting the Hh pathway could induce autoph-agy in HL-60 cells. The results showed that the combination ofvismodegib with CQ could also significantly inhibit cell viabilityof HL-60 cells (Fig. S5B). Although CQ could significantlyenhance the inhibition of cell viability by vismodegib in HL-60cells compared with the vismodegib treatment alone, thisenhancement was relatively weak when compared with theincrease in the inhibition of cell viability by CQ in vismodegib-treated BCR-ABLC cells. Moreover, inhibiting autophagy using

CQ could not enhance vismodegib-induced apoptosis in HL-60cells (Fig. S5C). Taken together, these results demonstrated thatsimultaneously inhibiting the Hh pathway and autophagy couldsharply induce apoptosis in BCR-ABLC CML cells.

Simultaneous inhibition of the Hh pathway and autophagyalso inhibited the BCR-ABL pathway in BCR-ABLC CML cells

The expression of the BCR-ABL oncoprotein is responsiblefor the development of CML.1 New generations of TKIs includ-ing nilotinib, dasatinib, bosutinib, exhibit effective inhibitoryeffects on the BCR-ABL oncoprotein; however, these TKIs haveno effect on BCR-ABL with the T315I mutation.43 Given that

Figure 8. Simultaneous inhibition of the Hh pathway and autophagy can significantly inhibit cell viability of drug-resistant CML cells. (A) K562, BaF3-BCR-ABL, BaF3-BCR-ABLT315I, and BaF3-BCR-ABLY253F cells were treated with imatinib or cotreated with vismodegib and CQ for 48 h, cell viability was mea-sured by CCK-8 assay. Error bars represent SD from triplicates. A typical result from 3 independent experiments is presented. (B) Phase-contrast imagesof cells treated with 1 mM of inmatinib or the combination of vismodegib (20 mM) and CQ (10 mM). (C) K562 and BaF3-BCR-ABLT315I cells were treatedwith imatinib (1 mM) or vismodegib (20 mM) or CQ (10 mM) or the indicated combinations of these agents for 48 h, cell viability was measured usingCCK-8. Error bars represent SD from triplicates. A typical result from 3 independent experiments is presented. **P < 0.01. (D) Fresh PBMCs were treatedwith DMSO, vismodegib, CQ or the combination of vismodegib and CQ for 24 h, Cell viability was measured using CCK-8. Error bars represent SD fromtriplicates. A typical result from 3 independent experiments is presented. *P < 0.05 versus to the group of cells treated with DMSO.


the combination of vismodegib with CQ could potently induceimatinib-resistant CML cell death, we determined whether thiscombined therapy could influence the kinase activity of theBCR-ABL oncoprotein, particularly the T315I mutation ofBCR-ABL. The phospholation of BCR-ABL and its downstreamprotein STAT5 is widely used to assess the kinase activity of the

BCR-ABL oncoprotein, while inhibition of the kinase activity ofBCR-ABL is an important indicator representing the pharmaco-logically inhibitory activity of TKIs on CML.36,37,44 Westernblot showed that combination of vismodegib with CQ decreasedthe phosphorylation of BCR-ABL and its downstream proteinSTAT5 in both K562 and BaF3-BCR-ABLT315I cells, while

Figure 9. Simultaneous inhibition of the Hh pathway and autophagy significantly induced apoptosis in CML cells. (A) K562, BaF3-BCR-ABL, and BaF3-BCR-ABLT315I cells were cotreated with vismodegib (20 mM) and CQ (10 mM) for 12 h, 24 h, and 48 h. The expression levels of PARP, CASP9, CASP3 and ACTBwere analyzed by western blot. (B) K562, BaF3-BCR-ABL, and BaF3-BCR-ABLT315I cells were treated as described in (A), apoptosis was detected by ANXA5and PI staining and flow cytometry. The percentages of ANXA5C PtdIns¡ cells (apoptosis) and ANXA5C PIC cells (necrosis) are presented in bar charts.Error bars represent SD from triplicate. A typical result from 3 independent experiments is presented. *P < 0.05; **P < 0.01; ***P< 0.001.


imatinib did not exhibit similar effects in BaF3-BCR-ABLT315I

cells (Fig. 11A to C), suggesting that a combination of vismode-gib with CQ could inhibit the kinase activity of BCR-ABL. Col-lectively, these results indicated that simultaneously inhibitingthe Hh pathway and autophagy could inhibit the BCR-ABLpathway in BCR-ABLC CML cells.

Discussion

CML, which is becoming more intractable due to drug resis-tance, is one of the most prevalent hematological malignancies allover the world. The aberrant expression of the chimeric proteinBCR-ABL is well known to mediate the development of CML.1

Over the past decades, accumulating knowledge of the mecha-nism of BCR-ABL has led to the development of TKIs for thisdisease, which has changed the history of CML therapy.2 Evenso, the majority of patients with CML exhibits drug resistanceare likely to relapse within several years.3 Today, drug resistance

is becoming the biggest challenge of CML treatment. It is urgentto investigate new treatment strategies or combination therapiesfor CML.

Our current study suggested that simultaneously inhibitingthe Hh pathway and autophagy might be a promising combinedtherapy candidate for overcoming the drug resistance of CML.First, our results revealed that inhibiting the Hh pathway couldinduce autophagy in CML cells. Autophagy is a basic physiologi-cal process in eukaryotes that is closely related to a variety ofhuman disorders including neurodegenerative diseases, autoim-mune diseases, cardiovascular diseases, and cancer.45-47 In partic-ular, the relationship between autophagy and cancer treatmenthas become a research hot spot in the field of cancer research.48

Several clinical trials are ongoing to evaluate the potential ofenhancing the antitumor effect of clinical drugs by targetingautophagy.49-54 The Hh pathway plays key roles in embryonicpattern formation and adult tissue renewal and maintenance.4

Evidence shows that the Hh pathway regulates autophagy inDrosophila and in several tumor types.27-30 This study reports the

Figure 10. Apoptosis induced by simultaneous inhibition of the Hh pathway and autophagy can be partially rescued by z-VAD-fmk. (A) K562 and BaF3-BCR-ABLT315I cells were pretreated with z-VAD-fmk for 1 h, then cells were cotreated with vismodegib (20 mM) and CQ (10 mM) or imatinib (1 mM) for48 h. Cells were subjected to western blot to detect the protein levels of CASP3 and ACTB. (B) K562 and BaF3-BCR-ABLT315I cells were treated asdescribed in (A), cell viability was measured by CCK-8 assay. Error bars shown are SD from triplicates. A typical result from 3 independent experiments ispresented. **P < 0.01. (C) K562 and BaF3-BCR-ABLT315I cells were treated as described in (A), apoptosis (ANXA5C PtdIns¡ cells) was measured by flowcytometry. Error bars shown are SD from triplicates. A typical result from 3 independent experiments is presented. **P < 0.01.


relationship between the Hh pathway and autophagy in CML forthe first time.

Second, we investigated the role of autophagy induced byinhibition of the Hh pathway, on its effect in the reduction ofcell viability in CML cells. Although other groups have reportedthat Hh inhibitors such as cyclopamine can reduce the cell viabil-ity of CML cells,17 treating with 20 mM of vismodegib for 48 honly inhibited cell viability of CML cells to about 80% of controlin the current study. When CML cells were cotreated with vis-modegib (20 mM) and CQ (10 mM) for 48 h, the cell viabilitywas about 15% of control, while cells of control groups (treatedwith DMSO, vismodegib or CQ alone) exhibited relatively nor-mal cell growth. Apparently, this combined strategy exhibited apotent synergistic cell killing effect on CML cells.

Moreover, we focused on the effects of simultaneously inhibit-ing the Hh pathway and autophagy on drug resistant BCR-ABLC

cells. BaF3-BCR-ABLT315I and BaF3-BCR-ABLY253F cells wereinsensitive to imatinib treatment, while the combination of vis-modegib and CQ could nearly eliminate all these cells. Mean-while, our results showed that the combination of vismodegiband CQ had little cytotoxicity on PBMCs. Furthermore, Hhinhibition could not influence the self-renewal and function ofadult haematopoietic stem cells.55,56 CQ, used in clinical practicefor more than half a century for the treatment of malaria andautoimmune diseases, has been approved by FDA as an autoph-agy inhibitor for clinical applications. This body of evidence indi-cates that the combination of vismodegib and CQ might be arelatively safe option to solve the problem of CML drug

resistance. Even if inhibiting autophagy could significantlyenhance imatinib-induced K562 cells and primary CML stemcells death, this combination strategy could not kill drug-resistantCML cells.25 The combination of Hh pathway inhibitors withautophagy inhibitors might be an effective combined therapy toovercome CML drug resistance.

Apoptosis is a type of typical programmed cell death, whichthe majority of anticancer agents can induce. We next exploredwhether the combination of vismodegib and CQ could induceapoptosis in CML cells. Caspase family proteins are key regula-tors of canonical apoptosis. Our results demonstrated that thecombination of vismodegib and CQ could potently induce apo-ptosis but was partially dependent on caspase activation in CMLcells.

Evidence shows that an activated MTOR pathway can pro-mote GLI1 transcriptional activity and oncogenic function whichlead to Hh pathway activation.57 The MTOR pathway also playsa key role in autophagy. In general, inhibiting the MTOR path-way induces autophagy.40 Our results indicated that inhibitingthe Hh pathway induced autophagy and concomitantly downre-gulated the AKT-MTOR pathway. Moreover, the evidence indi-cated that both the Hh pathway and autophagy were closelyrelated in modulating the BCR-ABL oncoprotein.58 Our resultsindicated that the combination of vismodegib and CQ couldinhibit the kinase activity of the BCR-ABL oncoprotein. It seemsthat there are complicated mechanisms in the combination of vis-modegib and CQ-induced cell death of BCR-ABLC CML cells.Killing drug-sensitive or -resistant BCR-ABLC CML cells

Figure 11. Simultaneous inhibition of the Hh pathway and autophagy also inhibited the kinase activity of BCR-ABL oncoprotein in BCR-ABLC CML cells.K562 and BaF3-BCR-ABLT315I cells were cotreated with vismodegib (20 mM) and CQ (10 mM) for 48 h. (A) The protein levels of p-BCR-ABL and p-STAT5were analyzed by western blot. (B and C) The relative protein expression levels are shown in bar chart. Densitometric values were quantified using theImageJ software and normalized to control. The values of control were set to 1. Data are means § SD of 3 independent experiments.


through simultaneously inhibiting the Hh pathway and autoph-agy was an interesting finding. Potential molecular mechanismsunderlying this phenomenon need deeper investigation in thefuture.

It is consequential to mention that extensive academic andindustrial efforts have contributed to the ongoing clinical trials ofHh inhibitors, such as vismodegib (GDC-0449, Roche), saride-gib (IPI-926, Infinity), sonidegib (LDE225, Novartis), BMS-833923 (Bristol-Myers Squibb), PF04449913 (Pfizer), andLY2940680 (Eli Lilly), for the treatment of a variety of cancers.7

Moreover, CQ and HCQ have been approved by the FDA forautophagy inhibition in clinical use. Thus, developing a largenumber of possible therapeutic combinations will be feasible tosimultaneously target the Hh pathway and autophagy for CMLtreatment.

Taken together, the current study indicated that inhibition ofthe Hh pathway could induce autophagy in CML cells. Ourresults also demonstrated that combination of inhibitors of these2 pathways could potently induce cell death in drug-sensitive or-resistant CML cells, while a single agent alone had mild effectson these cancer cells. This study suggested that the combinedtherapy, targeting both the Hh signaling pathway and autophagy,might be an effective strategy to overcome CML drug resistance.

Materials and Methods

Cell lines and cultureThe BCR-ABL-expressing K562 cells and the BCR-ABL-non-

expressing HL-60 cells were obtained from the Cell Bank of Chi-nese Academy of Sciences, Shanghai Branch (Shanghai, China).BaF3 cells stably expressing wild-type (p210) or mutant forms ofBCR-ABL (T315I and Y253F) were generously provided byProf. Xiaoguang Chen (Department of Pharmacology, Instituteof Materia Medica, Chinese Academy of Medical Sciences &Peking Union Medical College, Beijing, China). All cells werepassaged for fewer than 6 mo (fewer than passage number 100)after receipt from cell bank or resuscitation. Human peripheralblood mononuclear cells (PBMCs) were purchased from Shang-hai Blood Center (Shanghai, China). Fresh PBMCs were sepa-rated from peripheral blood of healthy donors and subjected toexperiments within 24 h. All cells were cultured in RPMI-1640medium (Invitrogen, 21875–091) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, 10099–141), 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen, 15140–122) at 37�C in a humidified incubator with 5% CO2.

ReagentsThe Hh inhibitor vismodegib (GDC-0449) (Shanghai bio-

chempartner, 20120611) was dissolved in DMSO (40 mM) forstorage. Other reagents were purchased as follows: CQ (Sigma,C6628), Bafi A1 (Sigma, B1793), RevertAid First Strand cDNASynthesis Kit (Thermo Scientific, K1622), TRlzol Reagent (Invi-trogen, 15596026), and FastStart Universal SYBR Green Master(Rox) (Roche Diagnostics, 4913850001). Monoclonal antibodieswere purchased as follows: MAP1LC3B (Cell Signaling

Technology, 3868), ATG5 (Epitomics, 3167–1), ATG7 (CellSignaling Technology, 8558), SQSTM1 (Epitomics, 3340–1),GLI1 (Epitomics, 6588–1), CTNNB1 (abcam, ab32572),GSK3B (Cell Signaling Technology, 12456), p-GSK3B (Ser9)(Cell Signaling Technology, 5558), tubulin (Cell SignalingTechnology, 2148), ACTB (Cell Signaling Technology, 4970),CASP3 (Cell Signaling Technology, 9665), CASP9 (Cell Signal-ing Technology, 9502), MYC (Cell Signaling Technology,5605), CCND1 (Cell Signaling Technology, 2978), PARP1(Cell Signaling Technology, 9542), p-AKT (Thr308) (Cell Sig-naling Technology, 2965), p-AKT (Ser473) (Cell SignalingTechnology, 4058), p-RPS6KB1 (Thr389) (Cell Signaling Tech-nology, 9234), p-EIF4EBP1 (Thr37/46) (Cell Signaling Tech-nology, 2855), p-MTOR (Ser2448) (Cell Signaling Technology,2971), and BCR-ABL activity assay kit (contains p-BCR-ABL[Tyr245] and p-STAT5 [Tyr694]) (Cell Signaling Technology,5300). Secondary antibodies were peroxidase-conjugated Affini-Pure goat anti-mouse (Jackson ImmunoResearch Laboratories,103757) and anti-rabbit IgG (Jackson ImmunoResearch Labora-tories, 104122). All other reagents were purchased from Sigma(St Louis, MO, USA).

Cell viability assayCell viability was measured using Cell Counting Kit-8 (CCK-

8) (Dojin Laboratories, CK04), as previously described.59 Briefly,about 5,000 cells per well were seeded in 96-well plates and thenexposed to vismodegib or autophagy inhibitors at indicated con-centrations. These cells were incubated with CCK-8 solution for0.5 to 1 h at 37�C. The optical density (O.D.) was measured atan absorbance wavelength of 450 nm. Images of phase contrastmicroscope were obtained using Nikon ECLIPSE TS100 micros-copy (Nikon Corporation, Japan).

Flow cytometry analysisApoptosis was measured using the Annexin V (ANXA5)-

FITC and PI Apoptosis Detection Kit (BD Bioscience, 556547)according to the manufacturer’s instructions. Analysis was per-formed using a FACSCalibur flow cytometer (Becton–Dickin-son, Fullerton, CA). Mitochondrial membrane potential wasmonitored after cells were staining with JC-1 mitochondrialmembrane potential assay kit (Beyotime, C2006) as previouslydescribed.60 For apoptosis and mitochondrial membrane poten-tial analysis, 30,000 cells were analyzed per sample. Cells werestained with PtdIns (Beyotime, C1052) and cell cycle was ana-lyzed using flow cytometry as previously described.60 For cellcycle distribution analysis, 10,000 cells were analyzed per sample.

Western blot analysisWestern blot analysis was performed as previously

described.61 Breifly, cells were harvested and subjected to proteinextraction using Cell Lysis Buffer. Equal amounts of the extractedprotein were run on SDS-PAGE gels and followed by transfer-ring membranes, blocking membrane, and incubating with pri-mary and secondary antibodies. The signals were developedusing an enhanced chemiluminescent detection reagent.


Quantitative real-time PCRTotal RNA extraction, reverse transcription, and quantitative

RT-PCR were performed as previously described.60 The follow-ing primers were used: GLI1, forward: AGCGTGAGCCT-GAATCTGTG; reverse: CAGCATGTACTGGGCTTTGAA.PTCH, forward: CTCTGGAGCAGATTTCCAA GG; reverse:TGCCGCAGTTCTTTTGAATG. SMO, forward: TGCCAC-CAGAAGA ACAAGC, reverse: GGAGATCTCTGCCT-CAACCA. These primers were synthesized by Shanghai GenerayBiotech Co., Ltd. (Shanghai, China). Relative mRNA expressionwas estimated by normalization with GAPDH expression.

Small interfering RNA transfectionCells were transfected with 50 nM siRNA using Lipofectami-

neTM 2000 Transfection Reagent (Invitrogen, 11668) accordingto the manufacturer’s instructions. Human SMO (Q000006608–1-A), ATG5 (siG10726164423), and ATG7 (siB111124164552)siRNA, were purchased from Guangzhou RiboBio Co., Ltd. MouseAtg5 (siB081013135244), Atg7 (siB10108143630) siRNA, andnonsilencing scrambled control (SCR) siRNA (siNO5815122147–1–10) were purchased from Guangzhou RiboBio Co., Ltd.

Confocal immunofluorescenceCells were transiently transfected with 50 nM of eGFP-

MAP1LC3B plasmid (Addgene, 11546) using LipofectamineTM2000 Transfection Reagent (Invitrogen, 11668). After 24 htransfection, cells were treated with 20 mM of vismodegib for48 h. Green fluorescence was observed under confocal micros-copy (Carl Zeiss LSM710, Carl Zeiss, Germany). Green fluores-cent spots were counted using ImageJ software.

Transmission electron microscopyBCR-ABLC cells were incubated with 20 mM of vismodegib

for 24 h, then harvested and processed as described.62 Samples

were analyzed with a JEM 1230 transmission electron micro-scope (JEOL Co., Ltd., Japan). Micrographs were taken at 7,000or 20,000 magnification.

Statistical analysisGraphPad Prism 5 (GraphPad Software Inc.., San Diego, CA)

was used to perform statistical analysis. The results wereexpressed as means § standard deviations (SD). Comparisonswere performed using the Student t test (2-tailed). P-value <

0.05 was considered statistically significant.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We would like to appreciate the generosity of Prof. XiaoguangChen (Department of Pharmacology, Institute of MateriaMedica, Chinese Academy of Medical Sciences & Peking UnionMedical College, Beijing, China) for friendly providing BaF3cells expressed wild type (p210) and mutant forms of BCR-ABL(T315I and Y253F).

Funding

This study was supported by grants from National Key BasicResearch Program of China (2015CB931800, 2013CB932502)and Shanghai Science and Technology Funds (14431900200,13431900303).

Supplemental Material

Supplemental data for this article can be accessed on thepublisher’s website.

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