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Translational Cancer Mechanisms and Therapy

KRAS G12C NSCLC Models Are Sensitive toDirect Targeting of KRAS in Combination withPI3K InhibitionSandra Misale1,2, Jackson P. Fatherree1,2, Eliane Cortez1,2, Chendi Li1,2, Samantha Bilton1,2,Daria Timonina1,2, David T. Myers1,2, Dana Lee1,2, Maria Gomez-Caraballo1,2, Max Green-berg1,2, Varuna Nangia1,2, Patricia Greninger1,2, Regina K. Egan1,2, Joseph McClanaghan1,2,Giovanna T. Stein1,2, Ellen Murchie1,2, Patrick P. Zarrinkar3, Matthew R. Janes3,Lian-Sheng Li3, Yi Liu3,4, Aaron N. Hata1,2, and Cyril H. Benes1,2

Abstract

Purpose: KRAS-mutant lung cancers have been recalci-trant to treatments including those targeting the MAPKpathway. Covalent inhibitors of KRAS p.G12C allele allowfor direct and specific inhibition of mutant KRAS in cancercells. However, as for other targeted therapies, the thera-peutic potential of these inhibitors can be impaired byintrinsic resistance mechanisms. Therefore, combinationstrategies are likely needed to improve efficacy.

Experimental Design: To identify strategies to maximallyleverage direct KRAS inhibition we defined the response of apanel of NSCLC models bearing the KRAS G12C–activatingmutation in vitro and in vivo. We used a second-generationKRAS G12C inhibitor, ARS1620 with improved bioavail-ability over the first generation. We analyzed KRAS down-stream effectors signaling to identify mechanismsunderlying differential response. To identify candidate com-bination strategies, we performed a high-throughput drug

screening across 112 drugs in combination with ARS1620.We validated the top hits in vitro and in vivo includingpatient-derived xenograft models.

Results: Response to direct KRAS G12C inhibition washeterogeneous across models. Adaptive resistance mechan-isms involving reactivation of MAPK pathway and failure toinduce PI3K–AKT pathway inactivation were identified aslikely resistance events. We identified several model-specificeffective combinations as well as a broad-sensitizing effect ofPI3K-AKT–mTOR pathway inhibitors. The G12CiþPI3Kicombination was effective in vitro and in vivo on modelsresistant to single-agent ARS1620 including patient-derivedxenografts models.

Conclusions: Our findings suggest that signaling adapta-tion can in some instances limit the efficacy of ARS1620 butcombination with PI3K inhibitors can overcome thisresistance.

IntroductionKRAS is among the most frequently mutated oncogenes in

human cancers. The most common mutations at codons 12 and13 lead to constitutive activation (GTP-bound state) through lossof intrinsic catalytic activity and/or loss of catalytic enhancementby GTPase-activating proteins (GAP) (1, 2). The development ofdirect inhibitors of KRAS has been very challenging and targetingof downstream effectors has proven largely ineffective (3). Recentbreakthrough work has identified KRAS-mutant allele-specific

inhibitors; these molecules are covalent inhibitors that bind tothe cysteine at position 12 of the G12C-mutant KRAS and are ableto downregulate KRAS downstream signaling (4–6).

Lung cancer is the leading cause of death by cancer in Westerncountries (7). While substantial advances have been made intreating genetically defined subtypes such as patients with EGFR-mutant or ALK-translocated lung cancer (8), an effective thera-peutic strategy for KRAS-mutant NSCLC, the most common(30%) genetically defined subtype, is still lacking. Mutationsleading to acquisition of a cysteine at codon 12 account for almost50% of patients with KRAS-mutant NSCLC (9); therefore, drugstargeting theG12Cvariant could have amajor therapeutic impact.

Pharmacologic targeting of a driver oncogenic event can yieldstrong responses and in some cases long lasting remissions (10),but their effectiveness is often limited by primary, adaptive, orsecondary resistance. Even within a given genetic and diseasecontext, resistance is often mediated by different molecularmechanisms, both genetic and not (11). For example, recentstudies have revealed the diversity of mechanisms of resistanceto tyrosine kinase inhibitors in lung cancer (12, 13). In addition,previous work showed that dependence on KRAS varies acrossKRAS-mutant models (14) and that even complete ablation ofKRAS using CRISPR-CAS9 does not always result in substantialloss of viability in KRAS-mutant models (15). Thus, we aimed to

1Massachusetts General Hospital Cancer Center, Boston, Massachusetts.2Department of Medicine, Harvard Medical School, Boston, Massachusetts.3Wellspring Biosciences, Inc., San Diego, California. 4Kura Oncology, Inc., SanDiego, California.

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

Corresponding Authors: Cyril H. Benes, Massachusetts General Hospital CancerCenter,149, 13th street, Charlestown, MA 02129. Phone: 617-724-3409; Fax: 617-643-5410; E-mail: [email protected]; Aaron N. Hata,[email protected]

doi: 10.1158/1078-0432.CCR-18-0368

�2018 American Association for Cancer Research.

ClinicalCancerResearch

Clin Cancer Res; 25(2) January 15, 2019796

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Published OnlineFirst October 16, 2018; DOI: 10.1158/1078-0432.CCR-18-0368

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evaluate the capacity of direct KRAS G12C pharmacologic target-ing to suppress viability in NSCLC models. In this report, wecharacterize the activity of ARS1620, a very recently reportedbioavailable inhibitor of KRAS G12C, on a series of NSCLCpreclinical models including PDXs. We show that KRAS G12C–mutantmodels display a range of sensitivity toARS1620. Throughdrug screening, mechanistic studies, and in vivo modeling, weidentify possible strategies towards the clinical application of thisnew class of drugs.

Materials and MethodsCell lines and compounds

Human lung adenocarcinoma lines CALU-1, HCC-44,HOP62,LU65, LU99A, NCIH1373, NCIH1792, NCIH2030, NCIH2122,NCIH23, NCIH358, and SW1573 were obtained from the Centerof Molecular Therapeutics at the MGH Cancer Center (Boston,MA). Short tandem repeat confirms identity of theCMTstocks andcell lines are not kept in culture for more than 2 months. Sangersequencing independently confirmed cell lines for this study toharbor the KRAS G12C mutation. Cells were cultured inRPMI1640 (Cellgro) supplemented with 5% FBS. Patient-derivedKRAS-mutant NSCLC cell lines MGH1088-1A and MGH1088-1Bwere established in our laboratory from malignant pleural effu-sion samples as described previously (16). TheMGH1062 cell linewas established from a resected brain metastasis. All patientssigned informed consent for an Institutional review board–approved protocol giving permission for research to be performedon their samples. MGH1062 patient received whole brain radi-ation; carboplatin/pemetrexed (2 cycles, no response); atezolizu-mab (response); docetaxel; navitoclax þ trametininb (4 cycles,stable disease); gemcitabine (1 cycle). Snapshot genetic analysisshowed KRAS G12C and TP53 E258�. MGH1088 patient did notreceive any treatments and died few weeks after the diagnosis.Snapshot genetic analysis revealed no other genetic lesionsother than KRAS G12C. After establishment, the presence ofthe clinically detected KRAS G12C mutation was verified bySanger sequencing. Patient-derived cell lines were maintainedin RPMI1640 supplemented with 10% FBS. All cell lines weremaintained in humidified incubators with 5% CO2 at 37�C.All drugs were purchased from Selleck Biochem except fromARS1620 (Araxes, Wellspring) and cetuximab (courtesy of MGHpharmacy).

Cell proliferation assaysFor short-term assays, cells were plated in 96-well plates at a

concentration of 6,000 (commercial cell lines) or 3,000 (patient-derived cell lines) cells perwell in 100mL/well of complete growthmedium and treated the following day with drugs diluted in

serum-free medium reaching a final concentration of 2.5% FBS.CellTiterGlo (Promega) reagent was added 96 hours after begin-ning treatment, and luminescence wasmeasured on a SpectramaxM5 spectrophotometer (Molecular Devices). All conditions weretested as triplicate biological replicates. All drug treatments wereadministered using a Tecan D300e Digital Dispenser. GI50 werecalculated using GraphPad Prism Software using the function log(inhibitor) versus response-variable slope (4 parameters). Valuesare capped at the maximum drug dose used.

Pull-down assayRas-GTP was pulled down using the Ras Activation Assay

Biochem Kit (Cytoskeleton) according to manufacturer's instruc-tions. Briefly, cells were lysed with cell lysis buffer and totalprotein was quantified using a BCA Protein Assay Kit (Pierce).Following quantification, 2mgof proteinwas incubatedwith Raf-RBD beads, rotating at 4�C for 2 hours. Bead–protein complexeswere collected and washed, before being resuspended in1� Laemmli buffer and boiled for 2 minutes at 95�C.

Antibodies and Western blottingAntibodies directed against the following were obtained from

Cell Signaling Technology and were used at a concentration of1:1,000 unless noted otherwise: p-Akt S473 (#9271), p-Akt T308(#4056), p-MEK1/2 S217/221 (#9154), p-p44/42 MAPK T202/204 (#9101), phospho-p70 S6Kinase T389 (#9205), totalMEK1/2 (#9122), total ERK1/2 (#9102), total AKT (#9272), total S6(#2117), total S6K (#9202), total RSK (#9355) and p-S6 S235/236 (1:2,000 dilution, #2211). KRAS (F234) antibody wasobtained from Santa Cruz Biotechnology and used at a concen-tration of 1:200. Antibodies against p-RSK1 p90 T359/S363(E238) and vinculin (EPR8185) were obtained from Abcam andused at a dilution of 1:1,000. Prior drug treatments, completemedia were replaced with 2.5% FBS media to parallel cell prolif-eration assays. Cells were washed with cold PBS and lysed in20mmol/L Tris (pH7.4), 150mmol/LNaCL, 1%NP-40, 1mmol/L EDTA, 1 mol/L EGTA, 10% glycerol, 5 mmol/L sodium pyro-phosphate, 50 mmol/L NaF, 10 mmol/L b-glycerol-P, 1 mmol/LNaVO3, 1 mmol/L PMSF, 0.5 mmol/L DTT, 4 mg/mL each of theprotease inhibitors leupeptin, pepstatin, and aprotinin. Lysateswere subjected to SDS-PAGE using 4%–12% Bis-Tris gels (Invi-trogen) followed by transfer to polyvinylidene difluoride mem-branes (PerkinElmer). Membranes were immunoblotted with theindicated antibodies and binding was detected with SuperSignalWest Femto Maximum Sensitivity Substrate (Thermo Scientific).

KRAS silencingThe siRNA-targeting reagents were purchased from Dharma-

con, as a SMARTpool of 4distinct siRNA species targeting differentsequences of KRAS transcript. Cell lines were grown and trans-fected with SMARTpool siRNAs using Dharmafect 4 (Dharma-con) following manufacturer's instructions. Cells were seeded in96-well plates at density of 6,000 cells/well in 100 mL of media.The day after, a final concentration of 1 mmol/L ARS1620,20 nmol/L SMARTpool siRNA or the combination of the 2 wasadded in 5 replicates (wells). Cell viability was assessed byCellTiterGlo (Promega) reagent 96 hours after beginning treat-ment, and luminescence was measured on a Spectramax M5spectrophotometer (Molecular Devices). Same concentration ofSMARTpool siRNA was used to assess KRAS knockdown byWestern blot analysis and RT-PCR.

Translational Relevance

KRAS G12C inhibitors could benefit a large group ofpatients with non–small cell lung cancer, but their clinicalpotential is still poorly defined. We show that ARS1620 is notalways effective as a single agent, suggesting the need for drugcombination strategies. Our studies show that resistance canbe overcome by PI3Ki combinations and might be useful tobroadly improve response across patients.

PI3K Targeting Overcomes Resistance to KRAS G12C Inhibitors

www.aacrjournals.org Clin Cancer Res; 25(2) January 15, 2019 797



http://clincancerres.aacrjournals.org/

qRT-PCRFollowing treatments, cells were lysed and RNA was collected

using theQiagen RNeasyMini Kit according to themanufacturer'sinstructions, including a DNase incubation to isolate RNA. TotalRNA was quantified using a Nanodrop 2000, and quality wasconfirmed on the basis of absorbance ratios of 260 nm/230 nmand 260 nm/280 nm. SuperScript II First-Strand Synthesis(Thermo Scientific) was used to generate cDNA. Quantitativereal-time PCR (qPCR) was performed using FastStart UniversalSYBR Green Master (Roche) on a LightCycler 480 PCR platform(Roche). HPRT, SDHA, and TBP were used as reference genes ineach reaction. Each data point is the average of 3 biologicalreplicates. DUSP6 forward primer: 50-CGACTGGAACGAGAA-TACGG-30, DUSP6 reverse primer: 50-TTGGAACTTACTGAAGC-CACCT-30; KRAS forward primer: 50-TGTTCACAAAGGTTTT-GTCTCC-30, KRAS reverse primer: 50- CCTTATAATAGTTT-CCATTGCCTTG-30; HPRT forward primer: 50-TCAGGCAG-TATAATCCAAAGATGGT-30, HPRT reverse primer: 50-AGTCTGGCTTATATCCAACACTTCG-30; SDHA forward primer:50-TGGGAACAAGAGGGCATCTG-30, SDHA reverse primer:50-CCACCACTGCATCAAATTCATG-30; TBP forward primer: 50-CACGAACCACGGCACTGATT-30, TBP reverse primer: 50-TTTTCTTGCTGCCAGTCTGGAC-30.

PI-AnnexinV stainingCells were treated for 72 hours prior to analyses. Cells were

washed in PBS then trypsinized. All material was collected,combined, centrifuged at 15,000 rpm for 5 minutes and washedwith PBS. Pellets were resuspended with 500-mL AnnexinVbinding buffer and stained with AnnexinV and propidiumiodide (PI). Analysis was performed on a BD Biosciences LSRIIflow cytometer. All presented apoptosis assays were performedin 3 biological replicates and the results represent averagesand SD.

High-throughput drug screeningHigh-throughput discovery of drugs able to sensitize cell lines

to ARS-1620 was performed essentially as described previously(16). Screening was performed in regular growth medium (highglucose), supplemented with 5% FBS. A panel of 112 drugs wasapplied to the cell lines at 9 different doses in the presence orabsence of ARS-1620 at afixed dose of 1mmol/L. Cell viabilitywasdetermined using CellTiterGlo after 5 days of drug treatment.Maximum dose was 10 mmol/L for all drugs except for trametinib(1 mmol/L) and phenformin (4 mmol/L) with a square root of10-fold dilution series (every other dose is 10-fold apart). Wecollected at least 2 biological replicates for each cell line andcombination. In the majority of cases we obtained 3 or morereplicates. For primary hit discovery, we identified synergisticcombinations based on shift in potency as single-agent versus incombination with ARS1620. Screen drugs were used at 9 differentdoses alone or in the presence of ARS1620. IC50 was calculatedusingDMSO (single agent) or ARS1620 alone (combination) as a100% relative viability control. IC50 was determined using drex-plorer in R. IC50 shift corresponds to the ratio of IC50s calculatedin the presence or absence of ARS1620. Primary hits were iden-tified using a 2-fold IC50 shift. We have previously shown that thisthreshold can translate into combination benefit in long-termassays in vitro and also in vivo (16). To further refine the list of hitswe used a 5� IC50 shift threshold. For each cell line, we then calleda consensus behavior for each drug: we eliminated hits that did

not reproducibly showa IC50 shift of 2�ormore in themajority ofreplicates (2 of 2 needed to be over 2-fold if only 2 replicates werepresent). This yielded a higher confidence list that is presentedin Fig. 3A were drugs have at least 1 instance of 5� shift and aconsistent behavior across replicates. Further confirmation ofsynergy was based on IC50 shift using experiments performedindependently. Furthermatrix dosingwas used to characterize thebehavior of the ARS1620 and PI3K inhibitor combination inselected models.

Establishment of in vivo models, patient-derived, and cell linexenografts

All mouse studies were conducted in accordance with theguidelines set forth by the Institutional Animal Care and UseCommittee of Massachusetts General Hospital. In vivo modelswere generated from commercial cell lines by subcutaneousinjection of cell suspensions containing 5 � 106 cells in 150-mLPBS into the flank of nudemice. Xenografts of the patient-derivedMGH1088 cell line were initially established in the same way andpropagated by surgical removal and reimplantation once thetumors reached 1,500mm3. TheMGH1062patient-derived xeno-graft model was established by direct implantation of surgicallyresected tumor tissue (brain metastasis) into the flank of an NSGmouse (Jackson Laboratories) followed by 2 rounds of serialpropagation in NSG mice; once established, the tumor waspropagated by serial transplantation in nude mice. For in vivodrug treatment studies, once tumors were established, mice wererandomized into 4 or 6 drug treatment groups, depending on theexperiment. All xenograftmodelswere treatedwithARS1620 (200mg/kg) and GDC-0941 (100 mg/kg), as single agents and incombination, in addition to a negative control vehicle treatment.H2122 xenografts were also treatedwith cetuximab (1mg/kg) as asingle agent and in combination with ARS1620. Drug treatmentswere administered 6 days a week by oral gavage (ARS1620 andGDC0941) or by intraperitoneal injections (cetuximab), andtumor volume was measured twice weekly along with miceweight monitoring.

ResultsWe collected 12 reported and commercially available NSCLC

lines harboring KRAS p.G12C mutation and validated the muta-tional status of KRAS. Sanger sequencing analysis of KRAS exon 2confirmed that 5 of the lines had G12C heterozygous mutationand 7 had a homozygous mutation (Fig. 1A). A number of othermutational events have previously been identified in systematiccell line sequencing efforts (COSMIC database, Cell Line Project).Specifically, for ERK/MAPK and PI3K pathway, among the 12lines 2 are KRAS amplified (LU65 and LU99A), 2 are PIK3CA-mutant lines (LU99A and SW1573), 1 is CRAF-mutant (HOP62)and 3 of the cell lines harbor mutations in receptor tyrosinekinases (RTK; NCIH1792, NCIH2122, NCIH23; SupplementaryTable S1). Thus, the genetic background of these models capturessome of the heterogeneity of NSCLCKRAS seen in tumors (17). Inthis study, we make use of ARS1620, a second-generation G12Ccovalent inhibitor with similar characteristics to the previouslyreported ARS853, but with higher on-target potency and superiorbioavailability (18). To initially estimate the impact of KRASG12C suppression in cells we measured the signaling impact ofARS1620 in vitro. Cells were treated for 48 hours with 1 mmol/LARS1620, a concentration known to completely inhibit KRAS

Misale et al.

Clin Cancer Res; 25(2) January 15, 2019 Clinical Cancer Research798




G12C in cells (18). Analysis of the phosphorylation status of keydownstream pathway nodes revealed that 2 of the initial 6 linestested displayed inactivation of ERK and only 1 displayed anyeffect on AKT phosphorylation. Interestingly, in the other 4 lines,there was either a minimal or no effect of the KRAS inhibitor ondownstream signaling after 48 hours of treatment (Fig. 1B). Wethen evaluated the impact of KRAS suppression on viability. Inkeeping with the signaling observations, dose–response prolifer-ation assays of the entire cell line panel revealed that only 2 of 12lines were highly sensitive to the direct KRAS targeting withlimited activity on the rest of the collection (Fig. 1C). Indeed,the only 2 sensitive cell lines were LU65 and NCIH358, whichboth displayed suppression of the ERK pathway at 48 hours (Fig.1B). Of note, in the current analyzed panel of G12C lines, G12Cisingle-agent activity was not predicted by KRAS G12C hetero- orhomozygosity or by the level of expression of KRAS, its ampli-fication status or other concomitantmutations on the RTK–PI3K–MAPK pathway (Fig. 1A and COSMIC database, Cell Line Project;Supplementary Table S1). This was somewhat surprising as KRASWTmight be expected to compensate for the inhibition of KRASp.G12C. In addition, the balance between KRAS WT versus mutant

has been shown to have an impact on the oncogenic activity ofKRAS mutant through dimerization or otherwise (19, 20). Thus,our results suggest that G12C zygosity status might not be apredictor of response to KRAS G12C–specific inhibitors inKRAS-mutant cancers. Comparison of the effect of G12C-specificinhibition with that of siRNA-mediated KRAS knockdown dem-onstrated high correlation (Supplementary Fig. S3), further sug-gesting that WT KRAS is not a major contributor to resistance inheterozygous models. To gain insight into the mechanistic basisof response or lack thereof, we determined the activation status ofKRAS and its downstream effectors following ARS1620 treatmentacross timeon the entire cell line panel.UsingARS1620at 1mmol/L, a time course revealed that GTP loading of KRAS was indeedblocked by the inhibitor and that inhibition was largely sustainedover time with none of the cells lines demonstrating substantialreloading of KRAS relative to basal pretreatment levels (Fig. 2A).MAPK pathway members, MEK1/2 and ERK1/2, were efficientlyinactivated in all cells lines initially (6 hours). However, thisinhibition was not consistently maintained across all models asseen in Fig. 1B. In the 2 most sensitive models LU65 andNCIH358, ERK phosphorylation was strongly suppressed

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Response to ARS1620 in KRAS G12C–mutant NSCLC cell lines. A, KRAS exon 2 Sanger sequencing results for the panel of 12 NSCLC cell lines; homozygous(homo), heterozygous (het). B, Signaling analysis upon ARS1620. The indicated cell lines were treated with 1 mmol/L ARS1620 for 48 hours and proteinlysates were used for Western blot analysis of KRAS downstream effectors. C, Dose response to ARS1620 after 96 hours treatment. Average and SD arerepresentative of at least 3 biological replicates quantified by measuring ATP content (CellTiterGlo assay).






Figure 2.

Analysis of RAS pathway inhibition upon ARS1620 treatment. A, Signaling analysis and KRAS-GTP pull down assay upon ARS1620 time course treatment,PC9 (KRAS wild-type) and A549 (KRAS G12S) cell lines are included as negative controls. B, Densitometry quantification, bands were quantified usingImageJ software and normalized on the loading control Vinculin. C, DUPS6 expression measured by real-time quantitative PCR. Cells were treated with 1 mmol/LARS1620 for the indicated times and the values are calculated on 3 housekeeper genes HPRT, SDHA, and TBP and normalized on untreated controls.D, Plot of GI50 values obtained for trametinib treatment (obtained from Supplementary Fig. S2) against GI50 values of ARS1620 (obtained from Fig. 1C).GI50 were calculated using GraphPad Prism software.

Misale et al.





throughout the 48-hour time course, while the third most sensi-tive model NCIH23 also displayed only minimal reactivation.However, in some other models, we observed rephosphorylationto a variable extent of MEK and ERK. Consistent with previouswork showing that MTORC1 repression is a good predictor ofviability outcome in response to growth factor pathway shutdown (21), S6 phosphorylationwas transiently reduced in severalmodels but this suppression was not sustained in the resistantones (Fig. 2A).

Interestingly, in a third category of models, ERK reactivationwas minimal but viability was still unaffected suggestingthat ERK activity is uncoupled from viability control in thesecells.

Overall, we observe a range of effects of KRAS G12C inhibitionon downstream signaling dynamics and the change in majordownstream signals observed are not sufficient to explain theviability outcome.While KRAS has been shown to activate PI3K atleast is some contexts, ARS1620-driven loss of KRAS-GTP resultedin suppression of p-AKT only in 3 of 12 cell lines studied (LU65,NCI-H358, and NCI-H23). Thus, we show that the PI3K pathway

is not under the sole control of mutant KRAS in KRAS G12CNSCLC models, and perhaps tumors, as shown previously incolorectal models (22).

Overall, these results suggest that KRAS does not uniformlycontrol prosurvival signals across KRAS G12C cell lines. Tomore finely resolve the dynamic signaling response throughoutthe pathway, we used densitometry quantification across thedifferent nodes measured. This further illustrated the differen-tial rebound of MEK/ERK phosphorylation contrasting with amuch more sustained and homogenous KRAS-GTP load loss(Fig. 2B; Supplementary Fig. S1). One key correlate of viabilityresponse in some cell lines appeared to be sustained suppres-sion of the MAPK pathway versus reactivation. To furtheranalyze the rebound in MAPK pathway activity, we measuredthe mRNA levels of DUSP6 upon ARS1620 treatment. DUSP6mRNA levels have been shown to more sensitively and accu-rately report the activation of the MAPK pathway comparedwith the phosphorylation status of ERK and MEK (23). DUPS6mRNA expression reported strong suppression of the MAPKpathway in all cell lines at 6 hours of treatment with ARS1620.

Figure 3.

High-throughput drug screen (HTDS) reveals ARS1620 and PI3K pathway inhibitors synergies and a heterogeneous pattern of RTK synergies. A, Pie chartof 112 drugs: color code corresponds to hit target type.B, Tophits identified across 112 drugs tested in combinationwithARS1620.Only combinations yielding at least 1instance of a 5-fold shift in IC50 in the presence of ARS1620 over single agent are presented. C, Pattern of a selected panel of recurrent effective combination acrosscell lines. IC50 shift (median across biological replicates) is shown.






In the ARS1620-sensitive models, reactivation was minimal,reaching only 10% of the initial levels at most. On the otherhand, in several of the resistant models, DUSP6 was robustlyreexpressed at 24–48 hours. Overall, the qPCR results werehighly consistent with the Western blot results (Fig. 2C). Thus,in a subset of KRAS G12C models, inhibition can lead to alargely sustained shutdown of the MAPK but this does notalways translate to a loss of viability.

To further understand this observation and the dependence ofthe KRAS cell lines on the MAPK pathway activity, we treated thecells with the potent MEK inhibitor trametinib and comparedthe viability outcome to the one obtained with ARS1620. Com-parison of GI50 values derived from ARS1620 or trametinibtreatments revealed 3 subcategories of models: (1) double sen-sitive models affected by ARS1620 and trametinib, (2) singlesensitive (ARS1620-resistant and MEKi-sensitive) models, whereMAPK rebound appears to mediate resistance, and (3) MAPKpathway inhibition impervious models with no effects on cellviability upon either direct KRAS or MEK inhibition (doubleresistant; Fig. 2D; Supplementary Fig. S2). Overall, these resultssuggest that the second category of models are resistant to

ARS1620 due to ERK pathway reactivation while the third isresistant because the ERK pathway does not exercise strongcontrol on viability.

Surprisingly, based on the large number of KRAS effectorsthat are implicated in proliferation and survival, we did notidentify cases of sensitivity to G12Ci with resistance to MEKi inthis panel of models. This initial characterization suggests thatsome G12C-mutant cells might be insensitive to direct KRASinhibition in at least 2 ways: by reactivating RAF–MEK–ERKaxis, or because the MAPK pathway is not solely controllingviability. Taken together, these results also show that althoughARS1620 and related inhibitors of KRAS p.G12C are efficientat suppressing KRAS activity, some tumors will likely be intrin-sically resistant to this class of drugs. These observationsare consistent with previous studies targeting KRAS genetically(14, 24). To further investigate the potential role of KRASreactivation in resistant models, we inhibited KRASG12Cwith ARS1620 and concomitantly knocked-down KRAS usingsiRNA (targeting both WT and G12C). KRAS knockdown effectswere very similar to G12C inhibition, as expected from ourcomparison with previous knockdown data (Supplementary

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Figure 4.

Validation of high-throughput drug screening. A, NCIH2122 dose response to RTK inhibitors alone or in combination with 1 mmol/L ARS1620 after 96-hourtreatment, averages and SDs are representative of at least 3 biological replicates quantified by measuring ATP content (CellTiterGlo assay). B, NCIH1792 doseresponse to RTK inhibitors alone or in combination with 1 mmol/L ARS1620 after 96-hour treatment, averages and SDs are representative of at least 3biological replicates quantified bymeasuring ATP content (CellTiterGlo assay).C, RTK inhibitors in combinationwith 1 mmol/L ARS1620 drug screening on "non hits"cell models. D, PI3K pathway inhibitors in combination with ARS1620 compared wirh FGFR inhibition (BGJ398) and EGFR inhibition (afatinib and cetuximab)in CALU-1 cell line. MK2206, AKT inhibitor; GDC0941, pan-PI3K inhibitor; alphaþbeta ¼ BYL719 (PI3Ka inhibitor) þ GSK2636771 (PI3Kb inhibitor). Results arerepresentative of at least 3 biological replicates quantified bymeasuringATP content (CellTiterGlo assay). Values obtainedwere then analyzedwith GraphPad Prismto obtain GI50 and calculate the GI50 fold change as follows: fold change (GI50) ¼ (GI50 single-agent drug)/(GI50 combination drug).

Misale et al.





Fig. S4). Although there was some small additive effect whencombining G12C inhibition with KRAS knockdown, this didnot yield viability suppression to the levels seen with MEKinhibition in NCI-H1792 cells that are sensitive to trametiniband only reduced viability to 40% in the double resistant cellline CALU-1 (Supplementary Fig. S4).

Because these KRAS G12C inhibitors are highly specific forthe mutant allele, they should provide a much better thera-peutic window than inhibitors of RAF, MEK, or ERK that act onnormal cells as well as cancer cells. Therefore, we speculatedthat drug combination involving G12C inhibitors couldachieve efficacy presumably with limited toxicity compared to,for example, combinations using MEK inhibitors. We thussought to identify pharmacologic combinations that couldovercome primary resistance to ARS1620. We performed ahigh-throughput combinatorial drug screening (16) to evaluatethe synergy of ARS1620 in combination with a panel of 112small molecules of high clinical relevance (Fig. 3A; Supple-mentary Fig. S5; Supplementary Data S1). To identify drugs ofinterest, we compared the sensitivity (IC50) of single agent tothe response in combination with ARS1620, at a concentrationyielding full suppression of KRAS GTP loading. Using IC50 shiftof over 2-fold as our primary hit definition metric, we foundthat among the 112 drugs, EGFR, FGFR, SFK, and PI3K pathwayinhibitors were the most synergistic with ARS1620 (Supple-mentary Figs. S3 and S5; Supplementary Data S2 and S3).BYL719 was seen as the drug most commonly synergizing withARS1620 across all cell lines. Other PI3K and AKT inhibitorswere also among the top hits. To further refine our hit list, weused a more stringent threshold of IC50 shift and determinedwhich drugs most consistently showed IC50 shift across bio-logical replicates (independent seeding dates). Drugs thatshowed at least 1 instance of 5-fold IC50 shift and consistentbehavior across replicates (majority rule) are shown in Fig. 3B.Behavior of selected individual drugs across cell lines is shownin Fig. 3C. The synergies observed with EGFR (lapatinib, gefi-tinib, BIBW2992) and FGFR (BGJ398, ponatinib) inhibitors areconsistent with recent results obtained with MEK inhibitorcombinations in KRAS-mutant cancers (25, 26). To furthervalidate these results, we used 2 cell lines with synergiesidentified for EGFR and FGFR inhibitors (NCIH2122 andNCIH1792, respectively). Using a panel of RTK inhibitors ina dose–response proliferation assay, we showed that the 2models displayed unique sensitivity to their specific hits incombination with ARS1620. This confirmed the drug screenresults showing a heterogeneous pattern of hits across cell lines(Fig. 4A and B). Indeed, while RTK inhibitor combinationsproduced some of the strongest synergies, these were not seenconsistently across models with the exception of ponatinib inthe high-throughput drug screen. Ponatinib is a potent butpromiscuous inhibitor of FGFR and the results with BGJ398, amore selective FGFR inhibitor, suggest that the observed syn-ergy may not be solely due to FGFR inhibition. These resultswere again in line with the previously published results usingMEK inhibitors in combination with different RTK inhibitors inKRAS NSCLC models showing that at least a subset of KRASNSCLC mutant are sensitive to FGFR þ MEK inhibition (27).Furthermore, combining ARS1620 with RTK inhibitors hadlimited impact in some of the double resistant models invalidation experiments (Fig. 4C) while combinations withPI3K–AKT–mTOR–targeted drugs showed increased efficacy

(Fig. 4D). Thus, each specific RTK inhibitor combination withKRAS G12C inhibitor might be of limited efficacy across KRASG12C patients. Furthermore, because no obvious genetic events(FGFR or EGFR amplification or activating mutations) under-lined the synergies observed, this suggested that it might bechallenging to identify patients that could benefit from a givenRTK inhibitor–based combination. Previous studies haveshown that PI3K suppression can be combined with MEKsuppression to yield in vivo efficacy against KRAS tumors(28, 29). Unfortunately, the combination of MEK and PI3Kinhibitors is very poorly tolerated in patients (30, 31). BecauseG12C targeting will be more tumor specific, we thereforeconsidered the potential for PI3K pathway inhibitors to sensi-tize KRAS G12C lines to ARS1620. While BYL719 was seen assomewhat more prominently synergistic with ARS1620 thanthe pan-PI3K inhibitor GDC0941, we reasoned that a targetingall PI3K isoforms would be more likely to be broadly effective.We thus decided to prioritize studies with GDC0941.

To mechanistically characterize the combination screen find-ings, we performed signaling analyses. We used a pan-HERinhibitor afatinib or GDC0941 in combination with ARS1620.Although the afatinib þ ARS1620 combination was effective atblocking the phospho-ERK rebound, only the GDC0941þARS1620 combination was able to simultaneously downregulateboth phospho-AKT and phospho-S6 across models (Fig. 5A). Asmentioned before, previous studies have shown that sustainedshutdown of phospho-S6 is a good predictor of effective sup-pression of cell viability (21). As expected, both proliferation andcell death (PI-Annexin V staining) assays demonstrated that thecombination of ARS1620 andGDC0941 was broadly effective. Inkeepingwith our previous results, while someof the KRASG12C–mutant cell lines also responded to the afatinib þ ARS1620combination, this was less consistent than the effect seen withthe GDC0941 þ ARS1620 combination (Fig. 5B and C). Weperform more detailed drug combination experiments using adrug combination matrix across 5 doses of ARS1620 and 9 dosesof GDC0941. We observed good efficacy and substantial synergyin most models, including ARS1620-resistant models such asNCI-H2122 and double (Trametinib/ARS1620) resistant modelssuch as SW1573 (Fig. 5D and E). Notably, while MEK inhibitor assingle agentwasmore broadly effective across KRASG12Cmodelsthan ARS1620, addition of a PI3K inhibitor broadly sensitizedcells to ARS1620. Cell viability assays comparing ARS1620 þGDC0941 versus trametinib þ GDC0941 combinations showedsimilar effect of the 2 combination treatments across G12Cmodels (Supplementary Fig. S6A–S6B). These viability resultswere consistent with effects seen on signaling events (Supplemen-tary Fig. S6C–S6E). We then evaluated the potential for PI3Ki þG12Ci in vivousingNCIH2122andSW1573as xenograftsmodels.We further used the anti-EGFR mAb cetuximab in combinationwith ARS1620 as a comparative treatment. Similar to the in vitroresults, NCIH2122 xenograft showed resistance to KRAS inhibi-tion alone. Notably, the combination with cetuximab resultedin tumor stabilization but only for a very limited amount of time.In contrast, the ARS1620 þ GDC0941 combination resulted inprolonged tumor stabilization (Fig. 5F–G). Further supportingthe potential for this combination, in SW1573 xenografts, treat-ment with ARS1620 þ GDC0941 also resulted in tumor regres-sion (Fig. 5F).

To further evaluate the efficacy of ARS1620, we generatedpatient-derived cell lines and xenografts from patients with KRAS






Figure 5.

ARS1620 þ PI3K inhibitor combination is effective in KRAS G12C–mutant models resistant to single-agent ARS-1620. A, KRAS pathway inhibition upon PI3Kior EGFRi combinations with ARS1620: ARS1620 (ARS), GDC0941 (GDC), and afatinib (AFA) were used at 1 mmol/L for the indicated times. Three ARS1620-resistant(HCC44, H2122 and SW1573) and 1 ARS1620-sensitive (LU65) cell lines are shown.B,Apoptosis measured in the 12 cells lines panel upon treatmentwith single agentsor drug combinations for 72 hours. ARS1620 (ARS), GDC0941 (GDC), and afatinib (AFA)were used at 1mmol/L and values are derived from the average of 3 biologicalreplicates. C, Single values of percentage of apoptotic induction in ARS1620-resistant cell lines. D, Drug combination validation experiments using ARS1620 andGDC0941: Heatmaps show viability through color-coding as percentage of cell viability normalized on untreated controls. E, Heatmaps of Bliss score for ARS1620-GDC0941 combination. F, NCIH2122 xenografts experiments with ARS1620 (200 mg/kg), GDC0941 (100 mg/kg) and cetuximab (1 mg/kg) plus combinations.SW1573 xenografts (G) were treated with the ARS1620 þ GDC0941 combination. Error bars represent SEM, 4 animals per group.

Misale et al.





G12C–mutant lung cancer. Three models (MGH1062-1A,MGH1088-1A, and MGH1088-1B) were generated from pleuraleffusions or surgical specimens of 3 different patients usingconditional reprogramming conditions as we described previous-ly (32). We treated these models in vivo with ARS1620 andobserved that they were all resistant to single agent. In keepingwith our results in previously established cell lines, all patient-derived xenografts were, however, substantially more sensitiveto the combination of ARS1620 with GDC0941 over eitherdrug as a single agent (Fig. 6A–C). Importantly, all the in vivotreatments, including the combination, were well tolerated asshown by good stability of the weights of the treated animals(Supplementary Fig. S7).

DiscussionThe efficacy of molecular targeted therapies for cancer treat-

ment is often compromised by mechanisms of resistance due togenetic heterogeneity and various compensatory mechanisms.While the emergence of direct KRAS inhibitors represents along-awaited opportunity for a large number of patients, under-standing the factors underlying sensitivity and resistance to thisnew class of compounds is critical for patient benefit. In this work,we show that lung cancer cell lines and newly established tumor-derived models display a range of sensitivity to KRAS inhibition.Surprisingly, sensitivity and resistance was not predicted by KRASallele zygosity status, suggesting that lack of targeting of the WTallele is not a straightforward predictor of sensitivity. We alsoconsidered other genetic alterations such as concomitant muta-tions or copy number variations in genes involved in the RASpathway; however, none of these alterations adequately explainresistance to G12C covalent inhibitors. Further studies are nec-essary to address these aspects using more comprehensiveapproaches. However, it is reasonable to speculate that resistanceto RAS-direct targeting could mainly rely on nongenetic or adap-tivemechanisms. Past studies based on synthetic lethality systemsor genetic silencing of KRAS fail to be further validated in the clinicsuggesting a more complex scenario. Therefore, our findingssupport previous studies that suggest a functional, clinicallyimpactful heterogeneity of KRAS-mutant cancers.

Interestingly, our data indicate that the adaptive response todirect KRAS inhibition is not always similar to MEK inhibitionconsistent with our previous work (24). Previous studies evi-

denced that RTK feedback activation is among the mainmechanisms of adaptive resistance to MEK inhibitors andtherefore vertical combinations with RTK inhibitors or RAFinhibitors are the most viable options. We found that RTKblockade was similarly effective in combination with KRASG12C inhibition in a subset of models with a similar hetero-geneity previously reported with MEK inhibitor plus RTK com-binations (27). However, our study suggests that combiningKRAS blockade with PI3K pathway inhibition is likely to bemore broadly effective. The mechanistic basis for this broadefficacy might differ across models. In some cases, PI3K acti-vation in response to MEK/ERK inhibition could participate inreactivation of the ERK pathway through adaptor proteins suchas GABs that bind PIP3. On the other hand, phospho-AKT isseldom affected by ARS1620 across the models tested, suggest-ing that the added benefit from PI3K inhibition might be due toconcomitant shutdown of 2 major interconnected pathwaysregulating proliferation and survival in carcinomas. Indeed,combining MEK and PI3K inhibition affects viability of manycancer models with diverse MAPK pathway–activating events(21, 28, 29, 33).

Clinical trials based on MEK inhibition failed in patients withlung cancer, at least in part due to low tolerability (28, 30, 31, 34).CombiningMEK andPI3K inhibitors is accompaniedwith furthertoxicity likely precluding proper shutdown of both pathways incancer cells and is unlikely to be further pursued clinically(35, 36). The use of the mutant-specific KRAS G12C drug isexpected to allow for a large therapeutic window.

PI3K inhibitors recently showed good efficacy as single agentsin some contexts and acceptable toxicities (37, 38). We thusbelieve that clinical development of G12C inhibitors wouldbenefit from combination with PI3K pathway inhibitor upfrontto maximize the response rate and reduce the development ofadaptive resistance mechanisms.

Altogether, our data show that KRAS G12C covalent inhibitorsrepresent a promising therapeutic opportunity for KRAS G12Clung cancers with optimal potential in the combination setting.

Disclosure of Potential Conflicts of InterestP.P. Zarrinkar, M.R. Janes, and L.-S. Li are employees of Wellspring Bios-

ciences, Inc., and have ownership interests (including patents) in Araxes PharmaLLC, which holds the patent rights to ARS-1620. Y. Liu has ownership interests(including patents) in Araxes Pharma LLC,which holds the patent rights to ARS-

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Figure 6.

KRAS G12C lung cancer patient-derived xenografts (PDXs) are sensitive to ARS1620 þ GDC0941 combination. MGH1088-1A (A), MGH1088-1B (B), andMGH1062 (C) patient-derived xenografts were treated with vehicle alone, ARS1620 (200 mg/kg), GDC0941 (100 mg/kg), and the combination of the 2drugs at the same doses. Error bars represent SEM and P values were calculated as t test with Mann–Whitney correction, 4 animals per group.






1620 and was an employee of Kura Oncology at the time of the study andthroughout the review process. A.N. Hata reports receiving commercial researchsupport from Amgen, Novartis and Relay Therapeutics. C.H. Benes is a con-sultant/advisory board member for Araxes and reports receiving commercialresearch grants from Amgen, Araxes and Novartis. No potential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: S. Misale, C. Li, A.N. Hata, C.H. BenesDevelopment of methodology: S. Misale, J.P. Fatherree, D. Lee, G.T. Stein,A.N. HataAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Misale, J.P. Fatherree, E. Cortez, C. Li, S.J. Bilton,D. Timonina, M. Gomez-Caraballo, M. Greenberg, V. Nangia, R.K. Egan,J.D. McClanaghan, G.T. Stein, E. Murchie, A.N. HataAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S.Misale, J.P. Fatherree, E. Cortez, J.D.McClanaghan,Y. Liu, A.N. Hata, C.H. BenesWriting, review, and/or revision of the manuscript: S. Misale, J.P. Fatherree,E. Cortez, C. Li, P.P. Zarrinkar, M.R. Janes, Y. Liu, A.N. Hata, C.H. BenesAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S.Misale, D. Timonina, P.Greninger,M.R. Janes,L.-S. Li

Study supervision: S. Misale, A.N. Hata, C.H. BenesOther (input and advice during progression of the study): P.P. ZarrinkarOther (synthesized KRAS G12C inhibitor ARS-1620): L.-S. Li

AcknowledgmentsThe authors thank Professor Maurizio Scaltriti for critical reading of the

manuscript. This work was supported by Stand Up To Cancer-American CancerSociety Lung Cancer Dream Team Translational Research Grant (SU2C-AACR-DT17-15). Stand Up to Cancer is a division of the Entertainment IndustryFoundation. Research grants are administered by the American Association forCancer Research, the Scientific Partner of SU2C. The high-throughput platformwas supported by a grant from the Wellcome Trust (102696). This work wassupported in part by a sponsored research agreement from Araxes that providedARS1620 and is developing inhibitors against KRAS.

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 February 15, 2018; revised July 23, 2018; acceptedOctober 11, 2018;published first October 16, 2018.

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2019;25:796-807. Published OnlineFirst October 16, 2018.Clin Cancer Res Sandra Misale, Jackson P. Fatherree, Eliane Cortez, et al. KRAS in Combination with PI3K InhibitionKRAS G12C NSCLC Models Are Sensitive to Direct Targeting of

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