Increased Synthesis of MCL-1 Protein Underlies Initial ...recently demonstrated that T790M can arise de novo during treatment; it follows that one potential therapeutic strategy to

Translational Cancer Mechanisms and Therapy

Increased Synthesis of MCL-1 Protein UnderliesInitial Survival of EGFR-Mutant Lung Cancerto EGFR Inhibitors and Provides a NovelDrug TargetKyung-A Song1,Yasuyuki Hosono2, Crystal Turner1, Sheeba Jacob1,Timothy L. Lochmann1,YoshikoMurakami3, NehaU.Patel1, JungohHam1, BinHu4, KristaM. Powell1,ColinM.Coon1,Brad E.Windle1, Yuko Oya2, Jennifer E. Koblinski4, Hisashi Harada1, Joel D. Leverson5,Andrew J. Souers5, AaronN. Hata6, Sosipatros Boikos7,Yasushi Yatabe3,8, Hiromichi Ebi2,8,and Anthony C. Faber1

Abstract

Purpose: EGFR inhibitors (EGFRi) are effective againstEGFR-mutant lung cancers. The efficacy of these drugs, how-ever, ismitigatedby the outgrowthof resistant cells,most oftendriven by a secondary acquiredmutation in EGFR, T790M. Werecently demonstrated that T790M can arise de novo duringtreatment; it follows that one potential therapeutic strategy tothwart resistance would be identifying and eliminating thesecells [referred to as drug-tolerant cells (DTC)] prior to acquir-ing secondary mutations like T790M.

Experimental Design: We have developed DTCs to EGFRiin EGFR-mutant lung cancer cell lines. Subsequent analyses ofDTCs included RNA-seq, high-content microscopy, and pro-tein translational assays. Based on these results, we tested theability of MCL-1 BH3 mimetics to combine with EGFR inhi-bitors to eliminate DTCs and shrink EGFR-mutant lung cancertumors in vivo.

Results: We demonstrate surviving EGFR-mutant lungcancer cells upregulate the antiapoptotic protein MCL-1 inresponse to short-term EGFRi treatment. Mechanistically,DTCs undergo a protein biosynthesis enrichment resultingin increased mTORC1-mediated mRNA translation ofMCL-1, revealing a novel mechanism in which lung cancercells adapt to short-term pressures of apoptosis-inducingkinase inhibitors. Moreover, MCL-1 is a key moleculegoverning the emergence of early EGFR-mutant DTCs toEGFRi, and we demonstrate it can be effectively cotargetedwith clinically emerging MCL-1 inhibitors both in vitro andin vivo.

Conclusions: Altogether, these data reveal that this noveltherapeutic combination may delay the acquisition of sec-ondary mutations, therefore prolonging therapy efficacy.Clin Cancer Res; 24(22); 5658–72. �2018 AACR.

IntroductionMetastatic EGFR-mutant non–small cell lung cancers (NSCLC)

have high response rates to EGFR inhibitors (EGFRi), in excess of

60% (1). Treatment with EGFRi has vastly improved the careof these patients, and advances in specificity of EGFRi towardmutated EGFR appear to improve these response rates evenfurther (2, 3).

However, responses to EGFRi are transient—�12 to 18monthsusually—as acquired resistance to these inhibitors continues to bethe main barrier to long-lasting responses (4). Acquisition ofresistance to the EGFRi gefitinib is often caused by a secondmutation in EGFR, T790M, which hinders the ability of gefitinibto inhibit EGFR (4). Studying EGFR-mutant cancer cell lines, werecently demonstrated that T790M can exist—even at hard-to-detect frequencies—prior to initiation of EGFRi treatment, oralternatively, appear de novo (5).

In the latter scenario, it remains largely unknown how clonesfrom EGFR-mutant lung cancers treated with EGFRi and have nopreexisting T790M mutations but eventually acquire them orother secondary bypass track mutations like MET amplification(6), survive initial drug therapy. To this point, our knowledge ofthe events leading to the survival of a subpopulation of cells[deemed "persister cells" or "drug-tolerant cells" (DTC)] remainslimited. A better understanding of DTC survival capacity, andmechanisms supporting that capacity, could inform upfront drugtreatment to kill DTCs, delaying or possibly even eliminating

1Philips Institute for Oral Health Research, VCU School of Dentistry and MasseyCancer Center, Richmond, Virginia. 2Division of Molecular Therapeutics, AichiCancer Center Research Institute, Nagoya, Japan. 3Department of PathologyandMolecular Diagnostics, Aichi Cancer Center, Nagoya, Japan. 4Department ofPathology, VCU School of Medicine, Richmond, Virginia. 5AbbVie, NorthChicago, Illinois. 6Massachusetts General Hospital Cancer Center and Depart-ment of Medicine, Harvard Medical School, Boston, Massachusetts. 7Division ofHematology, Oncology and Palliative Care, Virginia Commonwealth University,Massey Cancer Center, Richmond, Virginia. 8Precision Medicine Center, AichiCancer Center, Nagoya, Japan.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Authors: Anthony C. Faber, Virginia Commonwealth University,Perkinson Building Room 4134, 1101 East Leigh Street, Richmond, VA 23298.Phone: 804-828-0841; Fax: 804-828-0150; E-mail: [email protected]; andHiromichi Ebi, 1-1, Kanokoden, Chikusa-Ku, Nagoya, Aichi 464-8681, Japan.Phone: 81-52-762-6111; Fax: 81-52-764-2972; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-18-0304

�2018 American Association for Cancer Research.

ClinicalCancerResearch

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the onset of acquired resistance. In fact, important studies havepreviously demonstrated distinct molecular characteristics ofDTCs within subpopulations of cancer cells, including engage-ment of the insulin growth factor (IGFR) pathway and epigeneticmodifications (7, 8), presenting survival advantages to differentdrugs.

Although these other studies have focused mostly on signalingcascades and epigenomic alterations, we and others have dem-onstrated that deficiencies in apoptosis can underlie intrinsicresistance to EGFRi (9–13). This begs the question as to whethera deficient apoptotic subpopulation may transiently enrich fol-lowing initiation of EGFRi therapy, which could survive in arelatively dormant state until the eventual acquisition of a sec-ondary T790M mutation/other bypass track mutations. Further-more, if these DTCs could be pharmaceutically targeted with anappropriate drug within the current coterie of targeted therapies,they could be eliminated and theoretically delay or prevent theacquisition of resistance.

Materials and MethodsCell lines

The cell lines used in this study were the EGFR-mutant NSCLCcell lines PC9, HCC4006, and HCC827 and have been rigorouslycharacterized (5, 9, 13). These cell lines were cultured in RPMI1640 (Lonza)with 10% fetal bovine serumplus 1%penicillin andstreptomycin (Gibco). All the cell lines were routinely tested formycoplasma. In order to make the DTC lines, cells were seeded at adensity of 50% confluence in 100 mm3 dishes. Cells were treatedwith 50 nmol/L of gefitinib for 6 days, then the media werechanged, and cells were incubated for 3 days without gefitinib(Fig. 1C). Stable cell lines (GFP-IRES and GFP-IRES-MCL-1) wereproduced by transduction with pLENTI-GFP-IRES and pLENTI-GFP-IRES-MCL-1 as previously published (14).

Reagents for in vitro/in vivo experimentsGefitinib, dinaciclib, and osimertinib (AZD9291) were from

AbMole Biosciences. A-1210477 was kindly provided by AbbVie.S63845 was purchased from Chemietek. mTOR inhibitors(AZD2014 and MLN0128) were from Selleckchem and AbMole,respectively. All drugs were dissolved in DMSO at the stockconcentration of 10 mmol/L for in vitro experiments.

Western blottingLysates were separated on Nu PAGE 4% to 12% Bis-Tris midi

protein gel (Invitrogen). Polyvinylidene fluoride (PVDF) mem-branes were probed with antibodies against phospho-EGFR Tyr1068 (D7A5), pAKT Ser473 (D9E), MCL-1 (D35A5),

pMCL-1 Ser159/Thr163 (4579S), pERK1/2 Thr202/Thr204(D13.14.4E), NOXA (D8L7U), BCL-XL (C54H6), BCL-2(D55G8), cleaved PARP Asp214 (D64E10), pRpb1 CTD Ser2/5(D1G3K), pS6 Ribosomal Protein Ser240/244 (D68F8), mTOR(7C10), total 4E-BP1 (53H11), p4E-BP1 Thr37/46 (236B4), p4E-BP1 Ser65 (D9G1Q), c-MYC (D84C12), pp70S6K Thr389(108D2), cyclin D1 (2922S), and eIF4E (C46H6) from CellSignaling and eIF4G (A10) and GAPDH (6C5) from Santa CruzBiotechnology.

Bicistronic dual luciferase assayThe pFR_HCV_xbwas a gift fromPhil Sharp (Addgene plasmid

#11510; ref. 15). We transiently transfected the vector by lipo-fectamine 2000 (Thermo Fisher Scientific) for 48 hours. After 48hours, the cells were lysed by 1X passive lysis buffer for 20minutes, and luciferase activity was measured using the DualLuciferase Reporter Assay System (Promega, E1960).

Apoptosis and cell-cycle analysisFor apoptosis analysis, cells were plated to be�50% confluent

in 100mm3dishes. The next day, cellswere subjected to 3 cycles oftreatment with 50 nmol/L of gefitinib. Each cycle consisted oftreatment with drug for 3 days, assaying for apoptosis at 72 hours,followed by 2 days of no treatment. CyTM5 Annexin V andpropidium iodide (PI) staining solution were purchased fromBDPharmingen. After each cycle, FACSwas performed tomeasurethe presence of apoptotic cells on a Guava easyCyte(Millipore; Fig. 1A and B). For cell-cycle analysis (Fig. 4A), theindicated cells were exposed to drug for 24hours and then assayedas previously described (13).

siRNA and siRNA experimentsmTOR siRNAs (si mTOR) were purchased from Santa Cruz

Biotechnology (cat. #SC-35409) and Thermo Fisher Scientific(cat. #AM16708; assay ID: 145119 and #AM16708; assay ID:145120)). Scramble (si Sc) control siRNA was from Qiagen.HiPerFect transfection reagent (Qiagen) was used to produce theknockdown cells with 50 nmol/L of mTOR siRNAs following themanufacturer's protocol.

High-throughput cell content imagingFor the MCL-1 assay, we mixed either 10% of GFP control cells

or 10%of GFP-MCL-1 expressing cells with 90%of the parent cellline. The mixed cells were seeded at a low density (3 � 103 perwell) in two96-well blackplates (plates 1 and2).Oneof theplates(designated "P1")measured the percentage ofGFP among the cellpopulation at day 1 after seeding. The second plate (designated"P2") was treated as outlined in Fig. 1C, and then cells wereobserved under a high-throughput cell content imager (ImageXpress 6Micro XLS,Molecule Devices) 9 days later to determine ifthe population of GFP-MCL-1 cells increased compared withcontrol cells (Fig. 3B and C). DAPI (40,6-diamidino-2-phenylin-dole, 1:5,000) was used for counterstaining (SC-3598, Santa CruzBiotechnology). The fluorescence intensity of GFP within livingcells after drug treatment in each well using the multiwavelengthcell-scoring module was quantified on the imager using MetaX-press High-Content Image Acquisition and Analysis software.

Cell viability assayCells (3� 103 perwell) were seeded in 96-well black plates. The

next day, wemade various concentrations of drugs as indicated in

Translational Relevance

Our findings suggest that EGFR-mutant lung cancers surviveinitial targeted therapy through an increase in MCL-1 expres-sion. This is attributed to increased TORC1–eIF4E-driven cap-dependent mRNA translation of MCL-1. Importantly, MCL-1can be targeted pharmaceutically to improve responses. Thisstudy supports the use of MCL-1 inhibitors in combinationwith EGFR inhibitors to eliminate EGFR-mutant DTCs andpotentially delay the acquisition of EGFR inhibitor resistance.

MCL-1 Protects EGFR-Mutant Cancers from EGFR Inhibitors

www.aacrjournals.org Clin Cancer Res; 24(22) November 15, 2018 5659




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Figure 1.

MCL-1 is a survival factor that is markedly increased in DTCs and prevents apoptosis. A and B, Apoptosis analysis of EGFR-mutant NSCLC cell lines (A) PC9and (B) HCC827 cells. Cells were treated with 50 nmol/L of gefitinib. After PI/Annexin V staining, apoptotic cells were measured by FACS. Each cycle consisted ofdrug treatment for 72 hours (where FACS was performed) followed by no treatment for 48 hours before rechallenging with gefitinib. C, Schema ofgenerating gefitinib DTCs. During 6 days of drug treatment in duplicate plates (i.e., Par-1 and Par-2), cell viability decreased from 50% to <5%. After the media werechanged (day 7) to allow signaling to return to baseline (as would occur in patients' tumors), the cells regrew to >25% confluency, which represented theDTC population. Osimertinib-DTCs were made similarly, however not in duplicate. D, Lysates from EGFR-mutant NSCLC cell lines PC9, HCC4006, andHCC827 parental (Par) cells and the corresponding DTCs [gefitinib-DTCs (left) and osimertinib-DTCs (right)] were probedwith the indicated antibodies. E, PC9 cellswere treated as in Fig. C. The surviving cells (DTCs) were then assayed for relative MCL-1 mRNA expression (to ß-Actin) as determined by RT-qPCR. Theremaining cells were lysed and probed with MCL-1 and GAPDH antibodies, with the relative MCL-1 protein levels quantified. F, MCL-1 expression in EGFR-mutant cancers was evaluated by IHC on surgical resection of primary tumors before treatment and in EGFRi-treated tumors following confirmed partial responses.Error bars,�SD forA,B, and E, and the indicated data pointswere performed in quadruplicate forA andB, and in triplicate E. The data from E are technical replicates.�� , P < 0.001; ��, P < 0.01; NS ¼ not significant by Student t test.

Song et al.

Clin Cancer Res; 24(22) November 15, 2018 Clinical Cancer Research5660




the cell viability graphs through serial dilutions. We used theCellTiter-Glo luminescent cell viability assay (G7173, Promega)according to the manufacturer's protocol, with the exception thatwe added half of the reagent to each well that is recommended.

Chemotherapy treatmentPC9 and HCC4006 cells were seeded in 6-well plates at a

density of 3 � 105 cells/well. The next day, cells were treatedwith DMSO, cisplatin (10 mmol/L), or gemcitabine (500 nmol/L)for 24 h and 72 hours. After chemotherapy, the cells werereplenished with drug-free complete media in order to recoverfor 24 and 72 hours. Cells were then harvested and lysates wereprepared.

RadiotherapyPC9 and HCC4006 cells were seeded in a 6-well plate at a

density of 3� 105 cells/well. The next day, cells were exposed to 5gray (Gy) radiation. A nonradiated plate was simultaneouslymaintained as a control. After radiation treatment, the mediawere changed in both plates and the cells were incubated for anadditional 24 or 72 hours before harvesting.

Hematoxylin and eosin (H&E) and IHC stainingPatients with EGFR-mutant lung cancer were treated with

gefitinib. Following confirmation of partial response to the treat-ment, residual tumors were surgically resected. The stainings wereconducted using paired formalin-fixed, paraffin-embedded tis-sues before and after gefitinib treatment. Immunostaining withanti-MCL-1 (S-19,�100 dilution, Santa Cruz Biotechnology) wasfollowed by standard DAKO autostainer. The study was approvedby Institutional Review Board at Aichi Cancer Center and patientsprovided written informed consent. The analysis was conductedin accordance with Declaration of Helsinki.

Mouse studiesFor the experiments in Fig. 4D and E, we injected 3 � 106

PC9-GFP or PC9 GFP-MCL-1 cells with a 1:1 ratio of matrigelmatrix basementmembraneHC (Corning) subcutaneously in therear flank of Nod/SCID gamma (NSG) mice (6–8 weeks old,female). Drug treatment was started when the tumor reached�150 to 170mm3, andmice were randomized (n¼ 4–6mice pergroup). Tumors and bodyweight weremeasured 3 times per weekusing a digital caliper and electronic scale for the duration ofthe experiment. Tumor volumes were assessed by using length (L)�width (W)2� 0.52. Gefitinib was administered 4 days per weekby oral gavage at a concentration of 50 mg/kg/body weight in1% Tween 80 in sterile water.

For the experiment in Fig. 5G, we injected 3 � 106 PC9 cells(left) or 5 � 106 HCC827 cells (right) subcutaneously in bothflanks of NSG mice (6–8 weeks old, female, n ¼ 3–4 mice pergroup).When tumor reached�100 to 170mm3,we administeredgefitinib (50 mg/kg/body weight), S63845 (25 mg/kg/bodyweight), or combination treatment. The gefitinib group receivedtreatment by oral gavage daily for 3 consecutive days. The S63845group received treatment for 3 consecutive days by tail-veininjection in 25 mmol/L HCl, 20% hydroxypropyl-beta-cyclodex-trin (HPBCD). For the gefitinib and S63845 combination cohort,we administered S63845 first, then gefitinib 2 hours later for 3consecutive days. Tumors weremeasured every day using a digitalcaliper, and body weight was measured 3 times per week. For theexperiment in Fig. 6C, we injected 3 � 106 PC9 cells subcutane-

ously in NSG mice (6–8 weeks old, female, n ¼ 5–7 mice pergroup).Drug treatmentwas startedwhen the tumor reached�150to 170mm3. Tumors and body weight weremeasured 3 times perweek using a digital caliper and electronic scale for the duration ofthe experiment. Dinaciclib was administered by intraperitoneal(i.p.) injection at 20mg/kg/bodyweight twice per weekwith 20%HPBCD. The combination groupwas administered both gefitiniband dinaciclib in sequence on days when dinaciclib was admin-istered. The animal experiment was approved by the VirginiaCommonwealth University Institutional Animal Care and UseCommittee (IACUC protocol #AD10001048).

RT-qPCRTotal RNA was isolated with Quick-RNA Miniprep kit (Zymo

Research). cDNA was synthesized from 700 ng of total RNA bySuperScript III Reverse Transcriptase (Invitrogen) with Oligo (dT)primer (Ambion). RT-qPCR analysis was performed in triplicatewith SYBR Green master mix (Life Technologies) on a 7500 FastReal-time PCR system (Thermo Fisher Scientific) according to themanufacturer's protocol. The primer information is as follows: (i)MCL-1 (F) 50-GGGCAGGATTGTGACTCTCATT-30, (R) 50-GATG-CAGCTTTCTTGGTTTATGG-30, (ii) c-MYC (F) 50-AATGAAAA-GGCCCCCAAGGTAGTTATCC-30, (R) 50-GTCGTTTCCGCAA-CAAGTCCTCTTC-30, (iii) cyclin D1 (F) 50-TGAACTACCTG-GACCGCT-30, (R) 50-GCCTCTGGCATTTTGGAG-30, ß-actin (F)50-GGCATGGGTCAGAAGGATT-30, (R) 50-AGGATGCCTCTCTT-GCTCTG-30.

Statistical analysesFor Figs. 1A, B and E, 2D, 4C, 5A–G, 6B and C, P values were

determined by unpaired Student t test (��, P < 0.001; ��, P < 0.01;�, P < 0.05).

ResultsApoptosis is deficient in the DTC population

Wehypothesized that cells persisting after EGFRi exposureweredeficient in EGFRi-induced apoptosis, and as such, could consti-tute a DTC population that could be pharmaceutically targeted.To test this hypothesis, we treated EGFR-mutant PC9 andHCC827 NSCLCs with cyclical gefitinib. After each cycle, FACSwas performed to measure the presence of apoptotic cells. Wefound that both EGFR-mutant lung cancer cell lines were, asexpected (9, 14), vulnerable to gefitinib-induced apoptosis overa 72-hour period (i.e., cycle 1; Fig. 1AandB).However, reexposureto gefitinib over an additional 72-hour period was inadequate toinduce robust apoptosis in PC9 cells, and by the third cycleHCC827 cells also lost their ability to undergo apoptosis (Fig.1A and B). These data indicate a loss of apoptotic potential inearly-surviving EGFR-mutant lung cancer cells to EGFRi.

High MCL-1–expressing EGFR-mutant cells are enrichedfollowing short-term exposure to gefitinib, making up a DTCpopulation

To further assess the characteristics of early-formed DTCs, weexposed EGFR-mutant NSCLCs for 6 days with gefitinib, followedby replenishment with drug-free complete media for 3 days. Thereplenishment period served two purposes: (i) to allow baselinesignaling/expression to be studied in the DTCs, and (ii) to be ableto gather sufficient cellular material for study. The initial 6-daytreatment led to a cell confluency that empirically was�5%of the






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DTCs express highMCL-1 via cap-dependent translation.A,PC9-DTCs andparental PC9 cell lysateswere probedwith the indicated antibodies.B,ThreeEGFR-mutantlung cancer cell lines (PC9, HCC4006, and HCC827) were subjected to treatment with two separate pure mTORC inhibitors (1 mmol/L of AZD2014 or 200 nmol/L ofMLN0128) for 24 hours. C, PC9 cells were transfected with scramble (si Sc) or mTOR siRNA (si mTOR) for 48 hours and lysates were probed with theindicated antibodies.D, Expression of c-MYC (left) and cyclin D1 (right) at RNA (relative to ß-Actin) and protein levels (��, P < 0.01 by unpaired Student t test). E, PC9cells were transfected with a bicistronic construct measuring Firefly (cap-dependent translation) and Renilla (cap-independent translation, transfection control).Cells were harvested and measured using a luminometer. F, Total lysates treated as indicated (No Rx, no drug; WCL, whole-cell lysates) from parental andDTCswere subjected to immunoprecipitationwith an anti-MCL-1 antibody or an IgG isotype-matched control antibody. Ten percent of immunoprecipitated proteinswere probed with the indicated antibodies. G, Lysates were subjected to pull down with m7-GTP-Agarose beads (WCL, whole-cell lysates; percent is amount ofprotein blotted in relation to amount of protein immunoprecipitated). H, A model demonstrating cap-dependent translation is increased in DTCs throughincreased expression of cap-interacting proteins and mTOR, which results in upregulation of MCL-1. Error bars, �SD for D and E, and the indicated datapoints were performed in triplicate for D and in quadruplicate for E. �� , P < 0.01 by Student t test.

Song et al.





starting cell population; the subsequent 3 days without drugexposure allowed for modest regrowth of the cells (Fig. 1C). Wecollected these cells as well as the parental cells and performed

either RNA-seq to look at global gene-expression changes orWestern blots to analyze select critical proteins in the DTCs andparentals. In particular, because BCL-2 family members govern

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High-content cell imaging assay demonstrates enrichment of MCL-1–high cells. A, Schematic illustration of the experimental design using a high-contentcell imager (Par: 100% parental cell lines; Par þ GFP: 90% parental cell lines and 10% GFP expressing cell lines; Par þ GFP-MCL-1: 90% parental celllines and 10% GFP-MCL-1–expressing cell lines). The amount of GFP fluorescence was quantified in cells from Plate1 (P1) after 24 hours (day 1) from initialcell seeding. This was to confirm an equivalent (�10%) starting GFP-staining cell population for both the control arm (GFP) and the experimental arm (GFP-MCL-1).Plate2 (P2) was measured after day 10 in the same manner using the same settings. B, Representative images are shown at the indicated times andconditions. Scale bar, 150 mm. C, Bar graphs of surviving cells.






the cell death response to stress such as kinase inhibition, wefocused on the major BCL-2 family effector molecules (reviewedin ref. 16) in each of the EGFR-mutant lung cancer cell lines tested.Strikingly, we found expressionofMCL-1was sharply increased in

the bulk cell population of DTCs (Fig. 1D, left). Osimertinib(AZD9291; refs. 17, 18) is a third-generation EGFRi that targetsEGFR-mutant NSCLC, including those with T790M mutations,and may be used as first-line treatment (19). We also made DTCs

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MCL-1 prevents gefitinib-induced apoptosis in EGFR-mutant lung cancer cells and tumor regressions in EGFR-mutant lung cancer xenografts. A, Cell-cycleanalysis following 24 hours of drug treatment (50 nmol/L of gefitinib) compared with untreated (�) GFP expressing or GFP-MCL-1–expressing PC9 cells.B,PC9GFP-expressing orGFP-MCL-1–expressing cellswere left untreated (�) or treatedwith 50nmol/L of gefitinib for 24 hours, and lysateswere collected,Westernblotted, and probed with the indicated antibodies. C, PC9 GFP-expressing or GFP-MCL-1–expressing cells were treated with increasing amounts of gefitinibfor 72 hours and subjected to CellTiter-Glo. D and E, GFP-expressing or GFP-MCL-1–expressing PC9 cells (3 � 106) were injected into NSG mice. When tumorsreached approximately 150–200 mm3, mice were treated with gefitinib (50 mg/kg/body weight) for �30 days, and tumor volume was measured threetimes per week by electronic calipers. Average volume of tumors in each cohort �SD is shown. Control (black, �) and gefitnib treatment (red). F, Tumorsfrom PC9-GFP and PC9 GFP-MCL-1 were collected, Western blotted and probed with the indicated antibodies. Error bars are þSD for A and C; theindicated data points were performed in triplicate. Errors bars are þSD for D and E. �� , P < 0.001; �� , P < 0.01 by Student t test.

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The combination of gefitinib and MCL-1 inhibitors (A-1210477 or S63845) is effective to eliminate DTCs.A and B, EGFR-mutant NSCLC cell lines (PC9, HCC4006, andHCC827) were untreated (�) or exposed to 50 nmol/L of gefitinib, 10 mmol/L of A-1210477 (44), or the combination (gefitinib and A-1210477) and (A) cellviability was measured following 72 hours, (B) FACS apoptosis analysis following 24 hours was performed. C, PC9 cells were treated as in A and B but for 6 days indrugs and 3 days without drug, to recapitulate the DTC experiment (Fig. 1A), and at the end, cell viability was measured. D and E, EGFR-mutant NSCLC celllines (PC9, HCC4006, and HCC827) were exposed to 50 nmol/L of gefitinib, 1 mmol/L of S63845 (45), or the combination (gefitinib and S63845).D, Cell viability wasmeasured following 72 hours. E, FACS apoptosis analysis following 24 hours treatment was performed. F, PC9 cells were treated as inD and E but for 6 days in drugsand 3 days without drug, to recapitulate the DTC experiment (Fig. 1A), and at the end, cell viability was measured. G, PC9 and HCC827 cells were injected inboth flanks of NSG mice. Each cohort consisted of 3–4 mice. The mice were treated with gefitinib (50 mg/kg/body weight) and S63845 (25 mg/kg/body weight)three times, and tumor volume was measured everyday by electronic calipers for 13 days. Control (black), gefitinib (red), S63845 (blue) and combination (green).Waterfall plot of fold change at 8 days (PC9, left) or 11 days (HCC827, right) from initial tumor volume of individual mouse. Error bars areþSD for A, B, D, and E, andþSEM for C and F, and the indicated data points were performed in triplicate; �� , P < 0.001 by Student t test.






to osimertinib as we did with gefitinib, as depicted in Fig. 1C.Again, we found sharp upregulation of MCL-1 in the osimertinib-DTCs (Fig. 1D, right). In both sets of DTCs, other than BCL-2 inthe PC9 osimertinib-DTCs, other antiapoptotic BCL-2 familymembers were not upregulated, and, with the exception of NOXAin theHCC827gefitinib-DTCs, expressionof the keyproapoptoticBCL-2 family members BIM and NOXA was not markedly dimin-ished (Fig. 1D, left and right).

Although MCL-1 was upregulated over 8.2-fold by Westernblot, qPCR analysis showed a modest (1.9-fold) increase, indi-cating posttranscriptional induction (Fig. 1E). To determine if thisphenomenonwas also active in patients' specimens, we identifiedtwo EGFR-mutant lung cancer patients which we had pairedsamples before and after initial gefitinib treatment. The rebiopsywas taken prior to therapy resistance, but following a confirmedpartial response to getfitinib, thereforemimicking the in vitroDTCcells. Consistent with the in vitro data, expression of MCL-1 wasstrongly expressed in the post gefitinib-treated tumors comparedwith the gefitinib-na€�ve tumors (Fig. 1F). Altogether, these dataindicate a role for MCL-1 in EGFR-mutant DTC survival.

To better assess how MCL-1 was regulated in DTCs, we per-formedRNA-seq in the PC9 gefitinib-DTCs and PC9parental (i.e.,drug-na€�ve) cells. (The GEO accession number for these studies isGSE117610.) Consistent with the qPCR analysis, the RNA-seqdata reflected only a small increase inMCL-1mRNA expression inDTCs versus parental cells (Supplementary Table S1). MCL-1 isnotably controlled by mTORC1/eIF4E-mediated cap-dependenttranslation (20–23). Strikingly, mTOR and the cap-dependentinitiation factors eIF4G1, eIF4G3, and eIF3A (24) were four of themost significantly upregulated genes in theDTCs (SupplementaryTable S1). In cap-dependent translation, mTOR directly phos-phorylates the eIF4E-binding protein 1 (4E-BP1) at residues37/46, which primes 4E-BP1 for further phosphorylation at 65,all of which contribute to disassociation of 4E-BP1 from eIF4E,promoting the EIF4 complex and cap-dependent translation (22,23, 25). Consistently, the phosphorylated form of 4E-BP1 wassharply upregulated in the DTCs (higher migrating band; ref. 26),as indicated by Western blots probed with an antibody raisedagainst total 4E-BP1 and phospho-specific residues (Fig. 2A;Supplementary Fig. S1A).

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MCL-1 is a key target of EGFRi/dinaciclib combination therapy. Combination treatment with gefitinib (50 nmol/L) and the CDK9 inhibitor dinaciclib (100 nmol/L)for 24 hours followed by (A) Western blot analyses. B, PC9-GFP (G) and PC9 GFP-MCL-1 (M) cells were either left untreated (�) or treated with thecombination of gefitinib (50 nmol/L) and dinaciclib (100 nmol/L) for the indicated times and CellTiter-Glo cell viability assayswere performed. C, PC9 cells (3� 106)were injected subcutaneously into NSG mice. When tumors reached a volume of �150–170 mm3, mice were grouped in control (no drug, �), gefitinib(50mg/kg/bodyweight) byoral gavage for 4 timesperweek, dinaciclib (20mg/kg/bodyweight) by i.p. twice perweek, or the combination (gefitinib anddinaciclib).Waterfall plot of fold change from initial tumor volume. Please note 3 of 5 tumors in the combination group were completely regressed and undetectable(�� , P < 0.001; P value was tested by comparing gefitinib and combination treatment using Student t test; †, undetectable tumor). D, Schema demonstratinghow EGFR-mutant lung cancer DTCs survive early EGFRi therapy. For B and C, Student t tests were performed; �� , P < 0.001.

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We therefore hypothesized that DTCs had a greater capacity forthe biosynthesis of MCL-1 protein. As only some cancers down-regulate MCL-1 in response to cap-dependent translation disrup-tion (21), wefirst askedwhetherMCL-1was under cap-dependentregulation in EGFR-mutant lung cancer cells. We have previouslydemonstrated gefitinib treatment leads to downregulation ofmTORC1/p4E-BP1/MCL-1 in EGFR-mutant lung cancers (14)and confirmed osimertinib did the same (Supplementary Fig.S1B).Wemore directly probed themTORpathway by treating thePC9, HCC4006 and HCC827 cells with specific, pharmaceuticalinhibitors of TORC1/2. Here, we found that phosphorylation of4E-BP1was significantly inhibitedwith both TORC1/2 inhibitors,AZD2014 and MLN0128 (Fig. 2B). Consistently, MCL-1 levelswere decreased inbothAZD2014 andMLN0128-treated cells (Fig.2B). Furthermore, knockdown of mTOR with two differentsiRNAs also led to a sharp reduction in p4E-BP1 and MCL-1(Fig. 2C; Supplementary Fig. S1C). The combination ofMLN0128with gefitinib diminished cell viability more than either drugalone (Supplementary Fig. S1D) and increased apoptosis com-pared with either agent alone (Supplementary Fig. S1E) in paren-tal EGFR-mutant cell lines. In addition, the combination ofTORC1 inhibition and EGFR inhibition prevented the outgrowthof DTCs (Supplementary Fig. S1F).

We next investigated whether gefitinib/osimertinib survivingDTCs maintain MCL-1 expression under mTORC1 regulation.Affirming this pathway remained intact in the DTCs, phosphor-ylated 4E-BP1 and MCL-1 were inhibited in the DTCs followingeither AZD2014 or MLN0128 treatment, compared with controlDTCs (Supplementary Fig. S1G), and similar to the parental cells(Fig. 2B). To ensure T790Mwas not contributing to the DTC state,which could theoretically have enriched over the 6-day treatmentto confer resistance to gefitinib, we treated gefitinib-DTCs withosimertinib and found the DTCs were similarly resistant toosimertinib as they were to gefitinib, in stark contrast to thesensitivity of the parental cells (Supplementary Fig. S1H). Thesedata demonstrate the mTORC1/p4E-BP1/MCL-1 pathway isunder the control of the EGFR pathway in EGFR-mutant lungcancers, and the mTORC1/p4E-BP1/MCL-1 pathway is hyperac-tive in DTCs.

We next probed further the involvement of enhanced proteintranslation downstream of TORC1. Beyond MCL-1, other keyoncogenic proteins with short mRNA half-lives, prominentlyc-MYC and cyclin D1, are tightly regulated by TORC1-mediatedcap-dependent translation (27–29). Consistent with an enrich-ment of TORC1-mediated cap-dependent translation, bothc-MYC and cyclin D1 were sharply elevated at the protein levelin PC9 gefitinib-DTCs (Fig. 2A), with little change at the RNA level(Fig. 2D); upregulation of c-MYCand cyclinD1were confirmed inthe HCC827 gefitinib-DTCs (Supplementary Fig. S1A). We nextmeasured translation rates directly in DTCs, and compared themto parental cells. We used a dual luciferase reporter assay thatdetermined both rates of cap-dependent and cap-independent(IRES-dependent) translation, with the latter serving as a controlfor transfection efficiency (15, 30, 31). In this assay, Fireflyluciferase activity associateswith cap-dependent translationwhileRenilla luciferase activity associates with IRES-dependent transla-tional rates, and the ratio is reflective of cap-dependent rates. Wedetected a sharp increase in cap-dependent translation in theDTCs compared with the parental cells (�6-fold; Fig. 2E). Thesedata altogether indicate an increased cap-dependent translationin DTCs.

MCL-1 functions primarily as a binding partner to proa-poptotic BIM and BAK, by binding to and neutralizing them(16). Indeed, we and others have demonstrated BIM mediatesapoptosis in EGFR-mutant lung cancers treated with EGFRi(14, 32). In order to determine whether the increased MCL-1was functioning at this level, we immunoprecipitated MCL-1in the parental and DTC cells. As demonstrated in Fig. 2F,MCL-1 was bound to more BIM and BAK in the DTCs com-pared with the parental cells, indicating the increased MCL-1was neutralizing the two proapoptotic proteins. In addition,phosphorylated MCL-1 at S159/T163 is concomitantly upre-gulated in DTCs, further evidencing altered translation ascausative of increased MCL-1, and not dephosphorylation-driven stabilization (ref. 33; Fig. 2G). We next performedbinding assays with the 50-7 methylguanosine (m7G) capstructure, which is responsible for interacting with mRNAsundergoing cap-dependent translation (Fig. 2G). In fact,sharply increased levels of cellular eIF component proteins(eIF3A, eIF4G1, and eIF4G3) led to increased m7G cap:eIF4E:eIF4G binding complexes (�30-fold increase) in DTCs com-pared with parental cells (Fig. 2G and H), indicating increasedcap-dependent translation. In total, these data indicate DTCsupregulate the TORC1–EIF4 pathway, leading to an increasedrate and reliance toward translation of MCL-1. Furthermore,this MCL-1 binds BIM and BAK, which is important giventhat "free" BIM mediates EGFRi-mediated apoptosis in EGFR-mutant lung cancers (14, 34, 35).

Preexisting high MCL-1–expressing EGFR-mutant lung cancercells are enriched following short-term exposure to the EGFRigefitinib

As Western blot analyses can only clue us to an increase inMCL-1 expression within the whole-cell population, we soughtto more clearly determine whether a subpopulation of cells thatwere high in MCL-1 expression could enrich within the totalpopulation to survive as DTCs. To address this question, wedesigned a GFP-tagging assay on a high-throughput cell contentimager to determine whether preexisting, highMCL-1–expressingcells would enrich following initial gefitinib treatment,therefore recapitulating the increase in MCL-1 expression seenin the pooled cell population experiment (Fig. 3A and B) and theincrease in MCL-1 in the gefitinib-treated patient specimensfollowing partial responses (Fig. 1F). We utilized a bicistronicMCL-1-IRES vector tagged with GFP (GFP-IRES-MCL-1; ref. 36).We performed this experiment with a starting cell populationof approximately 10% of GFP and 10% of GFP-MCL-1 cells(Fig. 3A and B), and treated the cells as we did for the pooledcell population experiments (Fig. 1C). At the end of treatmentat day 7 followed by no drug for 3 days, the population ofGFP-MCL-1 cells at day 10 went from �10% to �60% inPC9 cells, and �10% to �30% in HCC4006 cells (Fig. 3C). Incontrast, the GFP control cells remained at similar levels (i.e.,10%) at day 10 (Fig. 3B andC).We also seeded untreatedGFP andGFP- MCL-1 as a control (Supplementary Fig. S2). As expected,both GFP and MCL-1–expressing cells with gefitinib treatmentundergo key signaling shutdown, as reflected by cell-cycle arrest(refs. 5, 9, 14; Fig. 4A). In contrast, the MCL-1 expressing cellsare protected from gefitinib-induced apoptosis (Fig. 4B), whichtranslates to large differences in viability across multiple dosesof gefitinib in EGFR-mutant NSCLCs (Fig. 4C). Altogether, thesedata indicate cells expressing high amounts of MCL-1 within an






EGFR-mutant lung cancer population of cells are refractoryto EGFRi-induced apoptosis, and high MCL-1 expression issufficient for EGFR-mutant cancer cells to persist followinggefitinib treatment.

MCL-1 prevents gefitinib-induced tumor regressionsWe next determined whether exogenous MCL-1 would

protect EGFR-mutant NSCLCs from gefitinib treatment in vivo.For these experiments, we injected PC9 tumors expressingexogenous MCL-1 or GFP-expressing control plasmids intoimmunocompromised mice. Consistent with the in vitro studies(Fig. 4B and C), the control tumors shrunk significantly(Fig. 4D), whereas the PC9 tumors expressing MCL-1 failedto shrink in response to gefitinib (Fig. 4E). These data indicateMCL-1 expression is sufficient to maintain the viability of PC9tumors (Fig. 4E and F).

MCL-1 is functional in the response of EGFRi in EGFR-mutantNSCLCs

MCL-1 is an experimentally proven drug target in severalcancers (37–40) and is an important oncogene downstream ofTORC1 (21, 25, 36, 41). Recently, MCL-1–specific inhibitorshave been developed. We first utilized the specific MCL-1inhibitor A-1210477 (39, 42) and treated the EGFR-mutantPC9 and HCC4006 cells with gefitinib in the presence orabsence of A-1210477. We found that the combination ofA-1210477 and gefitinib led to markedly fewer viable cellsthan either drug alone (Fig. 5A) and increased apoptosiscompared with either agent alone (Fig. 5B). Importantly, theaddition of A-1210477 to gefitinib in EGFR-mutant lung cancercells was sufficient to prevent the survival of most DTCs(Fig. 5C). Using a second MCL-1 inhibitor, with in vivo capa-bilities, S63845 (43), we reproduced these results in these threeassays (Fig. 5D–F). Lastly, lentiviral transduced shMCL-1 stablecells (Supplementary Fig. S3) greatly sensitized EGFR-mutantcells to increasing doses of either gefitinib or osimertinib,compared with scramble cells (Supplementary Fig. S3A). Thesedata altogether indicate MCL-1 inhibitors are sufficient toprevent the survival of most EGFR-mutant lung cancer DTCsthrough apoptosis, and suggest targeting MCL-1 in DTCs is apotent and rational drug strategy.

Cotargeting MCL-1 with S63845 and mutant EGFR withgefitinib is effective in vivo.

We next investigated whether the addition of the MCL-1i andEGFRiwas effective in vivo at improving initial responses to single-agent EGFRi. We treated EGFR-mutant tumors with gefitinib for 3days, S63845 for 3 days, or the combination for 3 days, andcompared these treatments to the control group.Measurements ofPC9 tumors at 8 days (Fig. 5G, left), similar to 6 (SupplementaryFig. S4A) and 7 days (Supplementary Fig. S4B), demonstratedtreatment with the combination of S63845 (25 mg/kg/bodyweight) and gefitinib (50 mg/kg/body weight) led to a robustshrinking of tumors, significantly exceeding what single-agentgefitinib was capable of (Fig. 5G). This was also demonstrated inHCC827 tumors at 11 (Fig. 5G, right), 9 (Supplementary Fig.S5A), and 10 days (Supplementary Fig. S5B). Importantly, thecombination was well tolerated (Supplementary Figs. S4C andS5C). These data indicate newer MCL-1 mimetics can combinewith EGFRi to substantially improve initial responses to EGFRiin vivo.

Cotreatment with the in-clinic CDK inhibitor dinaciclib iseffective through downregulation of MCL-1

Several pure MCL-1 inhibitors such as S63845 and AMG 176are now being clinically developed (39, 43), with Amgen's AMG176 in clinical trials (for example, clinical trial numberNCT02675452). Although it is not yet known whether thesedirect antagonizers of MCL-1 will demonstrate efficacy or toler-ability in humans, the cyclin-dependent kinase (CDK) inhibitordinaciclib has emerged as an effective MCL-1 inhibitor (44–46)that demonstrates tolerability and activity in clinical trials (47).We therefore assessed dinaciclib as a sensitizer to gefitinib inEGFR-mutant lung cancers to determine whether this strategywould also lead to the elimination of MCL-1 and suppression ofDTCs. Indeed, the combination led to potent downregulation ofMCL-1 and the induction of apoptosis in EGFR-mutant lungcancer cells to reasonably low doses of gefitinib (Fig. 6A).MCL-1 expression was sufficient to block the ability of thedinaciclib/EGFRi combination to induce apoptosis (Fig. 6B),translating to protection of total cell viability. In vivo, PC9 xeno-grafted tumors were treated with either single-agent gefitinib,single-agent dinaciclib, or the combination at the same doses.Single-agent dinaciclib had little effect on tumors, whereas, asexpected, single-agent gefitinib was able to block tumor growthandmodestly shrink tumors (Fig. 4D); however, the combinationwas sufficient to eliminate 60% of palpable tumors (Fig. 6C).These data further corroborate MCL-1 as a key early target inEGFR-mutant lung cancers (Fig. 6D) and demonstrate a secondclass of drug that may be used in combination with EGFRi toenhance early tumor responses.

MCL-1 is upregulated following different anticancer therapiesin EGFR-mutant NSCLC

We next asked whether MCL-1 was also upregulated in otheranticancer therapies, particularly ones that induced marked celldeath, in EGFR-mutantNSCLC. EGFR-mutantNSCLC lung cancercell lines were exposed to 10 mmol/L of cisplatin and 500 nmol/Lof gemcitabine for 24 hours (Supplementary Fig. S6A, left), ortreated for 72 hours and left to recover for an additional 72 hourswithout drug to enrich DTCs (Supplementary Fig. S6A, right).Although treatment with cisplatin led to the downregulation ofMCL-1 and cell death, as evidenced by cleavage of PARP(Supplementary Fig. S6A, left), the surviving fraction of cells(i.e., DTCs) had higher MCL-1 levels (Supplementary Fig. S6A,right). Similarly, MCL-1 levels were also higher in the gemcita-bine-surviving PC9cells ((Supplementary Fig. S6A, right).Wealsotreated the PC9 and HCC4006 cells with radiation and harvestedthe cells 24 hours and 72 hours later, respectively (SupplementaryFig. S6B). In the cells exposed to radiation, MCL-1 levels wereinitially decreased (Supplementary Fig. S6B, left). However, as theamount of cell death markedly increased in both the PC9 andHCC4006 cells over 72 hours, MCL-1 levels also increased in thesurviving populations treated with radiation, and in fact becamemarkedly higher in the surviving HCC4006 cells compared withuntreated cells. These data altogether indicate that MCL-1 enrich-ment among surviving cells following therapeutic cellular insultsappears widespread in EGFR-mutant NSCLC.

DiscussionIn this study, we demonstrate that (i) a subpopulation of EGFR-

mutant NSCLCs can endure initial EGFRi therapy, including both

Song et al.





first-generation and third-generation EGFRi, via prosurvivalMCL-1, (ii) the increase in MCL-1 expression seen in the bulkpopulation can be recapitulated by the enrichment of a smallpopulation of cells expressing exogenousMCL-1, (iii) the increasein MCL-1 occurs through an enrichment of TORC1–eIF4E-mediated cap-dependent translation of MCL-1, (iv) MCL-1 isfunctional and binds to BIM to neutralize it, and (iv) pharma-ceutical inhibition of MCL-1 can largely eliminate EGFR-mutantDTCs in vitro and improve initial responses in vivo.

The inability of EGFRi to induce apoptosis has been recentlydelineated as an important mechanism of upfront resistance toEGFRi (9–12, 48–50). Although these studies, including ours,have focusedon a clearly important role for the proapoptotic BIM,MCL-1 has become an increasingly relevant drug target andaccordingly the development of MCL-1–specific inhibitors hasexcelled (39, 43, 51). Of note, MCL-1 is an intimate partner ofBIM, serving as a key BIM neutralizer (17, 25). As further supportof an important role of MCL-1 in EGFR-mutant lung cancersurvival, and consistent withMCL-1 acting as a key survival factorin these cancers (Figs. 3 and4), it was recently reported thatMCL-1levels increase in EGFRi-acquired resistant patients (52). In addi-tion, recent studies have demonstrated a key role forMCL-1 in thesurvival of subsets of not only NSCLC (40), but also breast(42, 53), neuroblastoma (25), and blood cancers (43). Althoughthe protein levels of MCL-1 in DTCs were sharply upregulated(Fig. 1D), the transcript levels of MCL-1 from the same DTCs didnotmarkedly increase (Fig. 1E). Thiswas also true of the other twooncogenic proteinswithmarkedly short half-lives—cyclinD1 andc-MYC (29). Therefore, DTCs with high MCL-1 expression werelargely a result of protein changes, with subsequent studiesindicating this change was occurring at the level of translationaloutput. To put these data into context, while a disconnect betweentranscriptional outputs and translational outputs is well estab-lished in mammalian cells, interestingly, activation of the EGFRpathway reportedly confers a larger disassociation betweenthese outputs (54). Additionally, biosynthesis of much of thetranslational machinery itself is governed by translation (55),creating a self-regulating loop, at least somewhat liberated fromtranscriptional constraints. Biosynthesis of proteins in mam-malian cells are initiated through two major pathways: cap-dependent translation, where recognition of a modified gua-nosine to the 50 end of an mRNA by eIF4E is required (56), anda process independent of the eIF4 complex that relies oninternal ribosome entry sites (IRES). In cap-dependent trans-lation, the mTORC1 complex phosphorylates 4E-BP1, resultingin 4E-BP1 sequestration away from eIF4E, promoting thebinding of eIF4E to eIF4G and assembly of the eIF4F complex:in cancers in which this pathway is perpetually active, depen-dence develops on short-lived oncogenes, such as MCL-1(20, 23). In addition, phosphorylation of 4E-BP1 by themTORC1 complex is likely rate-limiting for cap-dependenttranslation in cancers (57). mTOR itself was highly upregulated(Supplementary Table S1; Fig. 2A), and 4E-BP1 was markedlymore phosphorylated, in the EGFR-mutant DTCs (as evidencedby higher migrating bands and/or phospho-specific antibodies;Fig. 2A; Supplementary Fig. S1A). When we measured cap-dependent translation directly, it was increased �6-fold in theDTCs (Fig. 2E), and MCL-1 was functional (Fig. 2F).

It is reasonably well established that cells, in response todifferent stresses, often downregulate global protein synthesis(58, 59), the bulk of which is orchestrated by cap-dependent

mRNA translation, therefore making our data on DTCs coun-terintuitive. However, eIF4, mTOR and pp70S6K signaling areamong the highest upregulated pathways in DTCs (Supple-mentary Table S2), corroborating the laboratory experiments inthis study (Figs. 1 and 2). It remains unclear if the cap-depen-dent translational machinery preferentially translates MCL-1and other proteins in DTCs, or the identity of the underlyingsignal to do so, if one such exists. Future studies will be aimedat delineating such things.

In this study, we found EGFR-mutant NSCLC DTCs surviveinitial EGFRi therapy by an orchestrated evasion of apoptosiscausedbya sharp increase inMCL-1mRNAtranslation. These dataare reminiscent of a previous study that demonstrated coincuba-tion with the BCL-2/xL inhibitor ABT-737 sensitized early-surviv-ing EGFR-mutant lung cancers to EGFRi (60). Our data pointingto a dependence onMCL-1 translation is not altogether surprisingbecause MCL-1 is highly regulated at the level of translation (61).Interestingly, while FBW7 depletion can lead to high levels ofMCL-1 in EGFR-mutant NSCLCs (52), in our DTC model thesecells did not have depleted FBW7 levels (Supplementary TableS3), indicating alternative strategies EGFR-mutant lung cancercells may use to increase MCL-1 levels. In addition, Wu andcolleagues recently found the p23-activated kinase (PAK1) couldactivate the PI3K pathway and upregulate MCL-1, contributing toEGFRi resistance (62); the same group also found the focal-adhesion–related protein paxillin could alter MCL-1 phosphor-ylation and expression, again contributing to EGFRi resistance(63). This study also further highlighted the important relation-ship between BIM andMCL-1 in EGFR-mutant lung cancers (63).Thefindings in these studies andoursmake a compelling case thatdifferent cellular processes can commonly lead to what seems tobe a critical signaling event in EGFRi resistance: MCL-1 upregula-tion. Of note, we also found evidence of increased KDM5Aexpression (Supplementary Table S1), verifying past reports onEGFR-mutant DTCs (7, 8).

There are nowmultiple MCL-1–specific inhibitors being devel-oped. As one of these drugs, AMG 176 (e.g., NCT02675452) iscurrently in clinical trial testing, the use of these drugs in com-bination with drugs such as EGFRi could be eventually tested.Importantly, we demonstrated in EGFR-mutant cell line xeno-grafts that S63845 markedly sensitized these tumors to earlygefitinib treatments (Fig. 5G).

We also demonstrate that dinaciclib, which has an establishedclinical tolerability profile and could be tested right away (47, 64),can serve as a surrogate MCL-1 inhibitor to thwart the emergenceof DTCs. Although dinaciclib also inhibits CDK1, CDK2, andCDK5, it has emerged as a potent MCL-1 inhibitor in vivo due tothe short half-life ofMCL-1mRNA that is sensitive to inhibition ofCDK9 (46, 65). Although dinaciclib has pleiotropic effects overlong-term treatments as a result of inhibiting the other CDKs, theemerging picture in people is much different. In a recent phar-macodynamics study of dinaciclib, it was demonstrated that itsexposure time peaks at 2 hours in humans (64). The same studydemonstrated dinaciclib effectively inhibited MCL-1 at 4-hourexposure, which returned to baseline levels at 24 hours. Impor-tantly, dinaciclib failed to inhibit pRB at bothof those timepoints,indicating it poorly inhibits CDK1/2 in humans (64). These datasuggest dinaciclib primarily acts through inhibitory effects onCDK9 in humans, without the exposure time to affect CDK1/2.This would indeed suggest one of dinaciclib's primary modes ofactivity is via downregulation of MCL-1 via CDK9, and therefore






would serve as a rational copartner with EGFRi to eliminate DTCsand improve efficacies of these drugs. Our in vivo data furthersupport the combination of EGFRi and dinaciclib as a viablecombination therapy to eliminate DTCs and enhance initialtumor responses (Fig. 6C).

Bhola and colleagues have recently identified a subpopula-tion of acute myeloid leukemia cells that are resistant toapoptosis (66). Impressively, the most apoptosis-resistant sub-populations are superior to predict response to chemotherapythan the overall apoptosis-primed state of the entire popula-tion, demonstrating a critical role of this subpopulation inchemotherapy response. This study also points to a phenom-enon in which DTCs emerge by circumventing apoptosis; inEGFR-mutant NSCLCs treated with EGFRi, the result is a sur-viving fraction of cells that may survive long enough to acquiresecondary and bypass track mutations and regrow in thepresence of drug (Fig. 6D). Eliminating these cells early couldthwart the development of acquired resistance in EGFR-mutantlung cancers and, as such, provide important insights into newpharmaceutical strategies that could improve responses toEGFRi in lung cancer.

Disclosure of Potential Conflicts of InterestJ.D. Leverson and A.J. Souers hold ownership interest (including patents) in

AbbVie. A.N. Hata reports receiving commercial research grants from Amgen,Novartis, and Relay Therapeutics. No potential conflicts of interest were dis-closed by the other authors.

Authors' ContributionsConception and design: K.-A. Song, A.N. Hata, H. Ebi, A.C. FaberDevelopment of methodology: J. Ham, A.C. Faber

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Hosono, S. Jacob, Y. Murakami, N.U. Patel, B. Hu,K.M. Powell, C.M. Coon, Y. Oya, J.E. Koblinski, Y. YatabeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K.-A. Song, C. Turner, T.L. Lochmann, Y. Murakami,B.E. Windle, J.D. Leverson, A.J. Souers, S. Boikos, Y. Yatabe, H. Ebi, A.C. FaberWriting, review, and/or revision of the manuscript: K.-A. Song, C. Turner,T.L. Lochmann, J.E. Koblinski, H. Harada, J.D. Leverson, A.J. Souers, A.N. Hata,S. Boikos, H. Ebi, A.C. FaberAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K.-A. Song, C. Turner, J. Ham, H. HaradaStudy supervision: K.-A. Song, A.C. FaberOther (management of In vivo work): K.-A. Song

AcknowledgmentsWe thank Katherine Borden (University of Montreal) and Cristian Bellodi

(Lund University) for helpful discussions. This work was supported by an NCIK22-CA175276 Career Development Award (A.C. Faber). A.C. Faber is sup-ported by the George and Lavinia Blick Research Fund and is a HarrisonEndowed Scholar in Cancer Research. Services and products in support of theresearch project were generated by the VCUMassey Cancer CenterMouseModelShared Resource, supported, in part, with funding fromNIH-NCI Cancer CenterSupport Grant P30CA016059. H. Ebi is supported by Grants-in-Aid for Scien-tific Research (16K07164) and Fund for the Promotion of Joint InternationalResearch (15KK0303) from Japan Society for the Promotion of Science. ThepFR_HCV_xb (Addgene plasmid #11510) was a gift from Phil Sharp.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 25, 2018; revised June 29, 2018; accepted August 1, 2018;published first August 7, 2018.

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2018;24:5658-5672. Published OnlineFirst August 7, 2018.Clin Cancer Res Kyung-A Song, Yasuyuki Hosono, Crystal Turner, et al. Novel Drug Target

-Mutant Lung Cancer to EGFR Inhibitors and Provides aEGFRIncreased Synthesis of MCL-1 Protein Underlies Initial Survival of

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