tumor intrinsic efﬁcacy by shp2 and rtk inhibitors in kras-mutant cancers · 2019. 11. 8. ·...

Cancer Biology and Translational Studies

Tumor Intrinsic Efficacy by SHP2 and RTKInhibitors in KRAS-Mutant CancersHuai-Xiang Hao1, Hongyun Wang1, Chen Liu1, Steven Kovats1, Roberto Velazquez1,Hengyu Lu1, Bhavesh Pant1, Matthew Shirley1, Matthew J. Meyer1, Minying Pu1,Joanne Lim2, Michael Fleming2, LeighAnn Alexander1, Ali Farsidjani1,Matthew J. LaMarche3, Susan Moody1, Serena J. Silver1, Giordano Caponigro1,Darrin D. Stuart1, Tinya J. Abrams1, Peter S. Hammerman1, Juliet Williams1,Jeffrey A. Engelman1, Silvia Goldoni1, and Morvarid Mohseni1

Abstract

KRAS, an oncogene mutated in nearly one third of humancancers, remains a pharmacologic challenge for direct inhibi-tion except for recent advances in selective inhibitors targetingthe G12C variant. Here, we report that selective inhibitionof the protein tyrosine phosphatase, SHP2, can impair theproliferation of KRAS-mutant cancer cells in vitro and in vivousing cell line xenografts and primary human tumors. In vitro,sensitivity of KRAS-mutant cells toward the allosteric SHP2inhibitor, SHP099, is not apparent when cells are grown onplastic in 2D monolayer, but is revealed when cells are grownas 3D multicellular spheroids. This antitumor activity is alsoobserved in vivo in mouse models. Interrogation of the MAPKpathway in SHP099-treatedKRAS-mutant cancermodels dem-onstrated similarmodulation of p-ERK andDUSP6 transcripts

in 2D, 3D, and in vivo, suggesting aMAPKpathway–dependentmechanism and possible non-MAPK pathway–dependentmechanisms in tumor cells or tumor microenvironment forthe in vivo efficacy. For the KRASG12C MIAPaCa-2 model, wedemonstrate that the efficacy is cancer cell intrinsic as there isminimal antiangiogenic activity by SHP099, and the effects ofSHP099 is recapitulatedbygenetic depletionof SHP2 in cancercells. Furthermore, we demonstrate that SHP099 efficacy inKRAS-mutant models can be recapitulated with RTK inhibi-tors, suggesting RTK activity is responsible for the SHP2activation. Taken together, these data reveal that manyKRAS-mutant cancers depend on upstream signaling fromRTK and SHP2, and provide a new therapeutic framework fortreating KRAS-mutant cancers with SHP2 inhibitors.

IntroductionProtein tyrosine phosphorylation plays a central role in

normal cell biology and pathogenesis of diseases includingcancer (1). A large number of tyrosine kinases and severalphosphatases are mutated in various types of cancer andfunction as oncogenes (2). The Src homology-2 (SH2)domain-containing phosphatase 2 (SHP2), encoded byPTPN11, was identified as the first protein tyrosine phospha-tase (PTP) oncogene (3). SHP2 contains two SH2 domains (N-SH2 and C-SH2), a PTP catalytic domain and a C-terminalproline-rich motif containing tyrosyl phosphorylation sites(Y542 and Y580; ref. 4). Under basal conditions, intramolec-

ular interactions between the N-SH2 and PTP domains impedesthe accessibility of substrates, thus suppressing the catalyticactivity of SHP2 (5). Upon RTK activation by upstream growthfactors or cytokines, the autoinhibitory interactions of SHP2are disrupted leading to its activation (6). In RTK-activatedcells (RTK, e.g., EGFR, FGFR, VEGFR) or KRAS-amplified cells,SHP2 transduces the upstream signal with its phosphataseactivity through guanine nucleotide exchange factors, includingSOS1 (4, 7). This in turn promotes activation of KRAS by GDPto GTP exchange, and subsequent activation of the RAS–MAPKpathway resulting in pro-proliferative and prosurvivalsignals (4, 6, 8, 9).

SHP2 has been shown to be important for many biologicalfunctions that are dependent on upstream growth factor signalingincluding angiogenesis and differentiation and proliferation oftissues (10–14). Inhibition of SHP2, using a selective allostericinhibitor, SHP099, has revealed therapeutic promise in cancersthat are dependent on RTKs (15–17). Upstream signaling fromRTKs, such as EGFR, canbe dispensable in cells with constitutivelyactive KRAS mutations, as evidenced by the lack of clinicalresponse to the anti-EGFR antibody, cetuximab, in KRAS-mutant colorectal cancer patients (18). However, not all KRASmutations impair intrinsic GTPase activity equally, and someKRAS-mutant cancers may still require robust upstream RTKactivity to maintain the GTP-loaded active state. Recent reportshave shown that the intrinsic hydrolysis of GTP to GDP inKRASG12C is higher than other KRASmutations, almost approach-ing that of wild-type KRAS (19–21) and that inhibition of SHP2

1Disease Area Oncology, Novartis Institute of Biomedical Research, Cambridge,Massachusetts. 2Exploratory Immuno-Oncology, Novartis Institute of Biomed-ical Research, Cambridge, Massachusetts. 3Global Discovery Chemistry, Novar-tis Institute of Biomedical Research, Cambridge, Massachusetts.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

H.-X. Hao and H. Wang contributed equally to this article.

Corresponding Author: Morvarid Mohseni, Novartis Institute of BiomedicalResearch, 250 Massachusetts Avenue, Cambridge, MA 02139. Phone: 617-871-3444; Fax: 617-871-4213; E-mail: [email protected]

Mol Cancer Ther 2019;XX:XX–XX

doi: 10.1158/1535-7163.MCT-19-0170

�2019 American Association for Cancer Research.

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can impact mutant KRAS signaling in genetically engineeredmouse models and in cancer cell lines (17, 22–24). In additionto SHP2's function of activating RAS downstreamof RTKs, there isgrowing, yet confounding evidence for an immunomodulatoryfunction of SHP2. SHP2 has been implicated in suppressing T-cellactivation by dephosphorylating PD1-recruited CD28 (25),although it has also been shown to be dispensable for T-cellexhaustion and for anti-tumor activity by PD1 blockade in CD4þ

T-cell specific knockout mouse models (26). Taken together,inhibition of SHP2 may exert antitumor effects in both tumorcell autonomous and nontumor cell autonomous manners.

In this report, we provide further evidence that inhibition ofSHP2 or RTKs is effective at treating a variety of KRAS mutantcancers that are dependent on upstream growth factor signaling,including some KRAS G13D and Q61H mutants.

Materials and MethodsCompounds

The following inhibitors were synthesized and structurallyverified by NMR/LC-MS at Novartis Institute of BiomedicalResearch (Supplementary Materials): SHP2 inhibitor/SHP099 (15, 16), MEK1/2 inhibitor/trametinib (27), pan-VEGF/PDGFR/FGFR inhibitor dovitinib (28), VEGFR2 inhib-itor/BFH772 (29), MET inhibitor/capmatinib (30), FGFR1-3inhibitor/BGJ398 (31), EGFR-inhibitor/erlotinib, HER2 andEGFR-inhibitor/lapatinib. Cetuximab antibody was producedby Eli Lilly (catalog # NDC-66733-958-23).

Cell linesCell lines evaluated were obtained from Novartis' CCLE col-

lection (32) and were tested to be free ofMycoplasma and murineviruses in the IMPACT1 PCR assay panel (RADIL, University ofMissouri, Columbia, MO). These cells were maintained in ATCCspecifiedmediumat 37�C in a humidified atmosphere containing5% carbon dioxide. In general, split ratios for subculturing con-sisted of 1:4 and 1:6 in T-225 flasks (Corning) and 1:4 and 1:6 in 3bilayer cell factories (Nunc Cell Factory) and then 1:4 in 10-layercell factories (Nunc Cell Factory). Cells were harvested at 85% to95% confluence by treating with 0.25% trypsin-EDTA (Gibco,catalog #14175-095), and centrifuged at 1,200RPM for 5minutesat 4�C.

Cell line engineeringMIA PaCa-2_SHP2-KO clone was generated by CRISPR tech-

nology as previously reported (33). MIA PaCa-2_SHP2-KO_SHP2T253M/Q257L cells were generated by infection of aboveMIA PaCa2_SHP2-KO clone with lenti-virus packaged using thepLKO-Trex-SBP-SHP2-T253M-Q257L plasmid (15) and selectionin TET-freemedia containing 1mg/mLG418.MIA PaCa-2_SHP2-shRNA cells were generated by infection of MIA PaCa-2 cells withretro-virus packaged using the pRSIUP-U6Tet-SHP2-shRNA-UbiC-TetRep-2A plasmid (34) and selection in TET-free mediacontaining 3 mg/mL puromycin.

In vivo xenograft efficacy studiesMicewere handled in accordance to the ILARGuide for theCare

and Use of Laboratory Animals in an AAALAC-accredited facility.Cell lines were confirmed to be free of mycoplasma and mouseviruses before use, cultured inmedium according to ATCC guide-lines then resuspended in ice-cold Hank's Balanced Salt Solution

(HBSS; Gibco, catalog # 25200-056) containing 50% Matrigel(Corning, catalog #354234). Athymic female nude mice wereinoculated subcutaneously into the right flank. Tumor-bearingmice were randomized into treatment groups once tumorvolumes reached approximately 250 mm3. For the dox-inducible SHP2-shRNA xenograftmodel,micewere fed ad-libitumwith chow containing doxycycline (Scott Pharma Solutions,catalog #1813539).

Tube formation assaysHUVEC cells were seeded into 6-well plates (0.2 � 106)

overnight in endothelial cell growth media (PromoCell, #C-22010) with 10% FBS. Following incubation, cells were treatedwith the respective compounds. After incubation, cells wereharvested and diluted to 0.2 � 105 cells/mL in endothelial cellbasalmedia (PromoCell C-22010) plus 10%FBS and 72 mL of thesuspension plus 8 mL of endothelial cell growth supplement(PromoCell C-39215) was plated within 96-well plates (Ibidi,#89646) coated with Matrigel (Millipore #ECM625). Brightfieldimages were captured (EVOS microscope, Thermo Fisher Scien-tific) 4 hours after incubation at 37�C, and tubes were quantifiedand analyzed using Image J (http://rsbweb.nih.gov/ij/).

Cell proliferation assaysFor 2D proliferation assays, 500 to 1,500 cells per well were

seeded into 96-well plates (Corning #3904) in 80 mL media perwell. For 3D proliferation assays, 3,000 to 4,500 cells per wellwere seeded into low attachment 96-well plates (Greiner Bio-one, #655976) in 80 mL media alone or with 2% or 20%Matrigel (Corning, #356237). Twenty-four hours after seeding,cells were treated with 20 mL compounds at different finalconcentrations as indicated. After 6 days of incubation, 100mL CellTiter-Glo reagent (Promega #G7573 for 2D; #G9683 for3D) was added to each well and cell viability was determinedaccording to the manufacturer's instruction. To determine theIC50 values, data were fitted using the dose response algorithmin GraphPad Prism as Y ¼ Bottom þ (Top-Bottom)/(1þ10 /̂((X-LogEC50))), in which Top and Bottom are plateaus in theunits of the y-axis and EC50 is the concentration of inhibitorthat gives a response half way between Bottom and Top. Fordetails on the soft agar colony formation methods and 2D and3D screen with SHP099 and the RTK inhibitor cocktail, pleasesee Supplementary Materials.

RNA preparation for RNA sequencingMIA PaCa-2 spheroids were generated by plating at 5,000 cells/

well of p-HEMA (Sigma, cat. # 192066) coated 384 microwellElplasia plate (Kuraray cat. # SQ 200 100 384) according to themanufacturer's protocol. For 2D samples, subconfluent cells wereseeded at 750 cells/well of a 384 well micro clear plate (Greinercat. # 781091). Next day, cells were treated with SHP099 at 20mmol/L, andDMSO control. At 3 and 19 hours posttreatment, 2Dand 3D cultured cells were lysed with 500 or 50 mL, respectively,per well of lysis buffer with DTT. RNA was extracted according tothemanufacturer's protocols within the RNeasy Kit (Qiagen cat. #74134). Quantification and quality of RNA assessed by theAgilent 2100 Bioanalyzer (Agilent cat. # G2938C): 2100 expertEukaryote Total RNA Nano Chip (Agilent cat. # 5067-1511). Fortranscriptome sequence and analysis details, please see Supple-mentary Materials. Gene expression datasets are available viaNCBI BioProject accession number PRJNA558508.

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ImmunoblottingThe following primary antibodies were used: phospho-ERK

(Cell Signaling Technology #4370), ERK (Cell Signaling Tech-nology #4695), phospho-RSK3 (Cell Signaling Technology#9348), phospho-AKT (Cell Signaling Technology #4060), AKT(Cell Signaling Technology #9272), p-SHP2 (Cell Signaling Tech-nology #3751), SHP2 (Cell Signaling Technology #3397), phos-pho-MET (Cell Signaling Technology #3077), phospho-STAT3(Cell Signaling Technology #9145), and tubulin (Cell SignalingTechnology #3873). Cells (2–7.5 � 105) in 2 mL growth mediawere seeded in 6-well plates (Corning, #3506 for 2D, #3471 for3D with ultralow binding). The 3D growth media contains 2%Matrigel (Corning, #356237). After one day (for 2D) or three days(for 3D, for spheroid formation), cells were treated with inhibi-tors at the indicated concentrations and duration. Cells were lysedon ice in RIPA buffer (Boston Bioproduct #BP-115) supplemen-ted with 1 mmol/L EDTA and Halt Protease and PhosphataseInhibitor Cocktail (Thermo Fisher Scientific #1861281). Lysateswere centrifuged at 14,000 rpm at 4�C for 15minutes and proteinconcentrations were determined using BCA protein assay(Thermo Fisher Scientific). Equal amount of protein was sepa-rated by electrophoresis in NuPAGE 4%–12% Bis-Tris gel(Thermo Fisher Scientific #WG1402BX10), and transferred tonitrocellulosemembranes (Bio-Rad, #1704159) for immunoblotwith indicated primary antibodies. The bound primary antibo-dies were visualized using goat anti-rabbit IgG (Hþ L) secondaryantibody conjugated with Alexa Fluor 700 and goat anti-mouseIgG secondary antibody conjugated with IRDye 800 CW andscanning with an Odyssey Infrared Imager System (Li-Cor).

Results3D cell culture reveals sensitivity to SHP2 inhibition inKRAS-mutant cancer cells

It was previously reported that cancer cell lines sensitive toKRAS or NRAS depletion were refractory to SHP2 depletion in apooled shRNA screen and RAS/RAF-mutant cell lines were notsensitive to SHP099 (15). The lack of sensitivity remains the casewhen KRAS mutation status is overlaid with sensitivity to eitherSHP2 (PTPN11) depletion in a pooled shRNA screen (Fig. 1A) orwith sensitivity to SHP099 assessed across cell lines and cancerlineages (Fig. 1B).

Interestingly, a small number of KRAS-mutant models, dis-played some sensitivity to SHP2 depletion by shRNA or inhibi-tionby SHP099 (Supplementary Tables S1–S3). Thesedata lightlysuggest that activatingmutants of KRASmay still be dependent onSHP2 or that other RAS isoforms (NRAS/HRAS or wild-type KRASfor heterozygotes)may contribute toMAPKpathway activation inthose cells. Cancer cells have displayed differential sensitivity totargeted anti-cancer agentswhen grown in three-dimensions (3D)compared with the two-dimensional (2D) setting; among theseare AKT/mTOR inhibitors (35, 36) and KRASG12C inhibitors (20).We therefore selected three KRAS G12 mutant cell lines that werenot sensitive to SHP099 in 2D culture (MIA PaCa-2/KRASG12C,KP4/KRASG12D, Capan-2/KRASG12V) and tested them in a 3Dculture setting with or without the addition of varying levels ofMatrigel in the same growth media as in 2D (Fig. 1C). Strikingly,MIA PaCa-2 cells displayed greater sensitivity to SHP099 whencultured as spheroids with an IC50 ¼ 0.39 mmol/L (0%Matrigel),contrary to their resistance in the 2D setting (IC50 > 30 mmol/L).Interestingly, the addition of either 2% or 20% Matrigel reduced

the SHP099 efficacy against MIA PaCa-2 3D spheroids. InKP4 cells, there was a greater than 10-fold reduction in IC50 forall 3D growth conditions (2D IC50 >30 mmol/L; 3D 0% MatrigelIC50¼1.42mmol/L; 3D2%Matrigel IC50¼ 2.97mmol/L; 3D20%Matrigel IC50 ¼ 3.49 mmol/L). In Capan-2 cells, which onlyformed spheroids with the addition of Matrigel, there was agreater than 10-fold reduction in IC50 in 3D (2D IC50 >30mmol/L; 3D 2% Matrigel IC50 ¼ 1.90 mmol/L; 3D 20% MatrigelIC50 ¼ 2.26 mmol/L). The 2D versus 3D sensitivity to the MEKinhibitor, trametinib, also displayed an IC50 shift in the samemanner as SHP099, with the exception of expected sensitivity totrametinib in 2D (IC50 ¼ 6.23–20.80 nmol/L; Fig. 1C). In con-trast, T3M-4 cells (Fig. 1D) bearing the KRASQ61H mutationremained insensitive to SHP099 (IC50 >30 mmol/L) even whencultured in 3D. To demonstrate that the SHP099 3D efficacyagainst KRAS G12–mutant cell lines is not a result of systematicsensitization to SHP099 from2Dto3Dgrowth,we also tested twoRAS/RAF wild-type EGFR dependent cancer cell lines that aresensitive to SHP099 in 2D, Detroit-562, and KYSE520 (Supple-mentary Fig. S1A). There was either no IC50 shift (Detroit-562) ora modest IC50 reduction (�3-fold, KYSE520) comparable withthe EGFR inhibitor erlotinib.

To determine whether the SHP099 sensitivity of KRAS-mutantcell lines in 3D is a general phenomenon and to ascertaincorrelations to specific KRAS-mutant alleles, we examined a largerKRAS-mutant cell line panel to evaluate SHP099 under both 2Dand 3D growth conditions (n ¼ 14, Fig. 1E; SupplementaryTable S4). For the majority of KRAS-G12–mutant cell lines (7/11) sensitivity to SHP099 in 3D was comparable with that of theKYSE520 in 2D or 3D. In summary, KRAS-mutant cancer cells,primarily those with G12 mutations, cultured as multicellularspheroids, were sensitive to SHP2 inhibition.

MAPK pathway is suppressed by SHP099 in KRAS-mutant cellsin both 2D and 3D culture

We next examined whether SHP099 had differential effects ontheMAPKand/or PI3Kpathways inKRAS-mutant cells cultured in2D versus 3D. The 3D growth condition used was 2%Matrigel forall cell lines to enable 3D growth of those cell lines requiringMatrigel, as 20% Matrigel is too rigid for cell harvesting. p-ERKand its downstream marker p-RSK3 was overall similarly inhib-ited by 3 and 10 mmol/L SHP099 at 2 hours of treatment, in allthree cell lines (MIA PaCa-2, KP4, and Capan-2) in both condi-tions, althoughnot as complete as the levels achievedby10nmol/L trametinib (Fig. 2A). In addition, rebound of p-ERK and p-RSK3at 24 hours following SHP099 treatment was similarly observedunder both conditions, despite the different antiproliferationeffects. Interestingly, the baseline levels of p-AKT, and to a lesserdegree for p-ERK, were lower when all three KRAS-mutant cellswere grown under 3D conditions. However, in T3M-4, baselinelevels of p-AKT in 3D conditions were also lower than in 2D,while p-ERK levels were comparable in 2D and 3D conditions(Supplementary Fig. S1B). It is also worth mentioning thatfluctuation of p-ERK levels was observed between experimentsand are likely a result of the size and how long the spheroidshave formed. Nonetheless, these baseline differences on p-ERKand p-AKT levels indicate differential wiring of cell signaling in2D versus 3D culture, which is further supported by theliterature (35, 37, 38). The combination of lower p-ERK levels,for SHP099 to inhibit, and a downregulated PI3K pathway,often implicated in cancer cell survival, may contribute to the

SHP2 Inhibition Is Effective at Treating KRAS-Mutant Cancers

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sensitivity to SHP2 inhibition in a subset of KRAS-mutant cellscultured in 3D.

Given that SHP099 suppressedMAPK signaling similarly in 2Dand 3D but had different impacts on cell proliferation and theacuteMAPK suppression is not complete, we utilized gene expres-sion analyses to determine whether alternative signaling path-waysmay contribute to thedifferential sensitivity to SHP099.MIAPaCa-2 cells, cultured in 2D or 3D and treated with SHP099 ortrametinib, were subjected to RNA sequencing (RNA-seq) anal-ysis. Using a recently described MAPK pathway signature withrelevance to clinical responses to MEK inhibition (MPAS: SPRY2,SPRY4, ETV5, DUSP4, DUSP6, CCND1, EPHA2, EPHA4; ref. 39)

along with several other MAPK pathway transcriptional markers(ETV1, DUSP5, DUSP7, EPHA7, BMF; ref. 40), we compared theeffects of SHP099 with those of trametinib. The MAPK pathwaytranscriptional effects of SHP099, while less than those of trame-tinib, did demonstrate MAPK pathway inhibition (Fig. 2B), con-sistent with the effects on p-ERK and p-RSK3 in the MIA PaCa-2cells (Fig. 2A). Interestingly, we also found that levels of SHP2transcripts at baseline in 3D were approximately 50% lower thanin 2D and were more comparable with those observed in tumorxenografts (Fig. 2C). Thedecreased SHP2 expression inMIAPaCa-2 was also confirmed at the protein level, concomitant withdecreased SHP2 phosphorylation at Y542, a marker of SHP2

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SHP099 inhibits MAPK pathway in KRAS-mutant cell lines cultured in 3D and 2D.A, Immunoblot of indicated proteins in MIA PaCa-2, KP4, and Capan-2 cellstreated with DMSO, SHP099 (3 or 10 mmol/L), or trametinib (10 nmol/L) for 2 hours and 24 hours in 2D and 3D culture (with 2%Matrigel), respectively. Tubulinserves as a protein loading control. 2D and 3D samples from each cell line were run in the same gel, and their signal intensities on the blot can be compared.B,MIA PaCa-2 cells cultured in both 2D and 3D were treated with trametinib or SHP099 for either 3 hours or 19 hours, followed by RNA extraction and RNA-seq.Duplicate samples were analyzed for MAPK pathway gene suppression or activation in response to treatment using a modified version of the MPASsignature (39). Heatmap depicts log2 fold change to the DMSO control group. Red depicts mRNA transcript upregulation and blue depicts transcriptdownregulation. C, PTPN11 gene expression levels by TPM (transcripts per million) of MIA PaCa-2 cells cultured in 2D, 3D, and xenografts derived from RNA-seqdata. Duplicate samples for 2D and 3D and expression data from two different xenografts in mice are shown. D, Immunoblot of p-SHP2(Y542) and SHP2 in MIAPaCa-2, Capan-2 cells cultured and treated as described in A.






activation by RTK activity (Fig. 2D; ref. 41). We then examinedSHP2 and p-SHP2 levels in the remainder of the cell lines andobserved a similar decrease of both SHP2 and p-SHP2 in Capan-2cells and a decrease only in p-SHP2 in KP4 cells and in T3M-4,when theywere cultured in 3D (Fig. 2D; Supplementary Fig. S1C).It is conceivable that the decreased levels of SHP2 or that SHP2activation in 3D may partially contribute to the increased sensi-tivity in 3D culture, as there is less SHP2 or less activated SHP2 toengage with the allosteric SHP2 inhibitor.

Deeper analysis of the transcriptional changes in the MIAPaCa-2 revealed substantial differences in the global geneexpression changes in response to treatment with SHP099 in3D culture as compared with 2D culture (SupplementaryFig. S1D) 19 hours after treatment. Gene ontology analy-sis (42, 43) revealed greater modulation of transcriptionalprograms after SHP099-treatment related to apoptosis, IFNgsignaling, cell cycle, and membrane localization of proteinsand is likely a reflection on the observed enhanced efficacy in3D. Interestingly, in 2D culture there are very few uniquelyaltered genes following SHP099 treatment, notably downregu-lation of genes associated with cell adhesion, p38 MAPK, andTGFb/SMAD signaling (Supplementary Fig. S1D); these samegenes display no differential expression with SHP099 treatmentin the 3D culture setting. In summary, KRAS-mutant cells aredependent on SHP2 for full MAPK pathway activation in both2D and 3D growth conditions. The efficacy of SHP2 inhibitionmay be more pronounced in KRAS-mutant cells in 3D growthconditions likely due to a variety of reasons including changesin MAPK pathway, the AKT pathway, and SHP2 expression andactivation.

In vivo efficacy and MAPK pathway suppression of SHP099 inKRAS-mutant models

We next evaluated whether SHP099 would be efficacious inKRAS-mutant human tumor xenograft models. In line with thein vitro 3D findings, SHP099, dosed at 100 mg/kg daily, wasefficacious in theMIA PaCa-2model, with equivalent response totrametinib (Fig. 3A). In contrast to the lack of in vitro response toSHP099 (Fig. 1D), the KRASQ61H T3M-4 tumors, displayed sen-sitivity to SHP099 in vivowith amodest improvement in responsewhen compared to trametinib (Fig. 3B).We next examinedMAPKpathway suppression in vivo as assessed by p-ERK and p-RSK3 inMIA PaCa-2 tumors. MAPK pathway suppression was observedwith SHP099 (Fig. 3C), consistent with the newly appreciatedbiology that the KRASG12C-mutant cancers retain intrinsic GTPaseactivity and are regulated by RTKs and SHP2 (17, 21–23). How-ever, in T3M-4 tumors (KRASQ61H), suppression of p-ERK bySHP099 was variable and modest among tumors, while trame-tinib robustly and consistently suppressed p-ERKwithout impact-ing p-RSK3, suggesting a decoupling of the p-RSK effect from ERKactivity in this cell line (Fig. 3D).

We extended the in vivo profiling of SHP099 across a panel ofKRAS-mutant xenograft models, including the KYSE520(KRASWT/EGFR-dependent) and RKO (BRAFV600E; Fig. 3E; Sup-plementary Fig. S2A–S2I). Relative to the KYSE520 (Supplemen-tary Fig. S2A), SHP099 achieved comparable antitumor activity intwo KRASG12C models, NCI-H358 and MIA PaCa-2, as well as aKRASG12Dmodel, KP4 (Fig. 3A andE; Supplementary Fig. S2B andS2C). In addition, tumor growth inhibition was observed in allother KRAS-mutant models tested (Supplementary Fig. S2D–S2G), except in NCI-H460 harboring homozygous KRASQ61K

mutations, where there was no effect of SHP099 treatment (Sup-plementary Fig. S2H), and more comparable with SHP099 treat-ment in the RKO BRAFV600E xenograft model (Fig. 3E; Supple-mentary Fig. S2I).

We next examined the extent of MAPK pathway inhibitionin vivo following SHP099 treatment using DUSP6 mRNA expres-sion, a downstream transcriptional readout for MAPK pathwayactivity, 3 hours after the last treatment dose (Fig. 3F).Overall, themagnitude of DUSP6 suppression correlated with the magnitudeof antitumor activity of SHP099. For the KRASG13D model,HCT15, and the KRASQ61H model, T3M-4, we observed onlymodest changes in DUSP6 suppression, which is likely insuffi-cient to cause the observedmagnitudeof antitumor activity. Thesedata further suggest that mechanisms outside of the MAPKpathway may also contribute to the in vivo efficacy of SHP099in KRAS mutant cancers.

Efficacy of SHP2 inhibition in KRAS-mutant cancer is tumorintrinsic

Several reports have demonstrated that VEGFR signalingdepends on SHP2 in endothelial cells and is required for angio-genesis (14, 44, 45). To ascertain whether the efficacy of SHP099in KRAS-mutant models in vivo was in part due to an antiangio-genic effect of VEGFR2 signaling inhibition, we evaluated SHP2inhibition in vitro in tube formation assays using human endo-thelial cells, HUVECs. SHP099was unable to inhibitHUVEC tubeformation in vitro at 5 mmol/L, a concentration above what can beachieved in vivo at theMTD of 100mg/kg, daily (Fig. 4A).We nextevaluated whether efficacy of SHP099 was due to antiangiogeniceffects in vivo. In contrast to dovitinib, a multi-targeted RTKinhibitor with antiangiogenic effects (46) that clearly causedtumor devascularization in a KRAS-mutant colorectal cancerhuman PTX model, HCOX4087, in vivo treatment with SHP099,had no gross impact on tumor vascularization and was qualita-tively comparable to the vehicle and trametinib-treated tumors(Fig. 4B; Supplementary Fig. S3A). We further examined in vivoefficacy of a selective VEGFR2 inhibitor, BFH722 (29), in MIAPaCa-2 and T3M-4 xenografts and found that it had minimalefficacy (Supplementary Fig. S3B and S3C), in contrast toSHP099, while also displaying inhibition of mouse CD31mRNAexpression (Fig. 4C), suggesting that these models are not signif-icantly dependent on tumor vascularization for in vivo growth.SHP099 also maintained efficacy when MIA PaCa-2 cells weregrown orthotopically by surgical implantation directly into thepancreas, demonstrating that the effects of SHP099 are unlikely tobe an artifact of subcutaneous xenografts exhibiting enhancedpermeability and retention (EPR; Supplementary Fig. S3D;ref. 47). Having established that SHP099 had little impact ontumor vascularization, we evaluated whether genetic depletion ofSHP2within KRAS-mutant tumor cells would replicate the effectsof SHP099. Using CRISPR-Cas9 technology, SHP2 knockout(crKO) MIA PaCa-2 cells were generated and confirmed(Fig. 4D). Knock out of SHP2 in the MIA PaCa-2 cell line hadonly modest growth effects when grown in a monolayer culture,whereas significant growth inhibitionwasobservedwhen the cellswere grown in 3D (Fig. 4D) or when implanted subcutaneouslyinto nude mice (Fig. 4E).

To assess the role of SHP2 in tumor maintenance versusinitiation and to also rule out potential clonal effects from theSHP2-crKO model, MIA PaCa-2 cells transduced with a doxycy-cline (dox)-inducible shRNA targeting PTPN11 was generated

Hao et al.





(Supplementary Fig. S3E). Comparable with SHP099 treatment,SHP2 knockdown resulted in significant antiproliferative effectsin a 3D colony formation assay (Supplementary Fig. S3F). Wenext performed a head-to-head comparison of efficacy betweenSHP099 and SHP2-shRNA using the engineered dox-inducibleSHP2-shRNAMIA PaCa-2 cells. Following tumor randomization,efficacy of SHP099 and dox-induced SHP2 knockdown wasevaluated for 15 days (Fig. 4F). Efficacy between SHP2 geneticdepletion and pharmacologic inhibition was comparable, with amodest but statistically insignificant improvementwith the inhib-itor treatment group, attributed to greater MAPK pathway sup-pression as measured by DUSP6, and incomplete depletion ofSHP2 in the dox-treated xenografts (Supplementary Fig. S3G). To

demonstrate that the effects observed by SHP099 are due to on-target SHP2 inhibition, MIA PaCa-2 SHP2-crKO cells were trans-duced with a dox-inducible SHP099-binding deficient mutant,SHP2T253M/Q257L, and in vitro 3D efficacy was evaluated in thepresence of dox and SHP099 (Fig. 4G). The phosphatase activityof SHP2T253M/Q257L mutant was demonstrated to be similar tothat of wild-type SHP2 (15) and the expression level ofSHP2T253M/Q257L mutant in the SHP2 KO background was verysimilar to the endogenous level of SHP2 in the parental cells.SHP099 was inactive in cells expressing the compound-bindingdeficient mutant of SHP2, demonstrating that the antiprolifera-tive activity of SHP099 is due to on target inhibition of SHP2(Fig. 4G).

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KRAS-mutant cancers are sensitive to SHP2 inhibition in vivo. Antitumor efficacy of SHP099 administered orally at the doses, schedules, and duration indicatedin MIA PaCa-2 (A) and T3M-4 (B) subcutaneous xenograft models. Data are plotted as the treatment mean� SEM (n¼ 5). Red dotted line represents tumorvolume at randomization. C and D, Immunoblot analysis of levels of p-ERKT202/Y204 and p-RSK in KRAS-mutant xenograft models, MIA Paca-2 and T3M-4, from Aand B, treated with SHP099 and trametinib 3 hours after the last dose. Protein loading amount was normalized and verified by tubulin loading control. Eachseparate column represents an individual treated tumor. E, Antitumor efficacy of SHP099 administered orally across a panel of in vivo xenograft cell line andprimary tumor models. Data plotted is the percent test/control for each model compared with its respective control group at the end of treatment� SEM (%T/C). Each model is color coded with respect to its KRAS/BRAF genetic status. In vivo efficacy data for each model are in Supplementary Fig. S2 along withcomparative efficacy to trametinib. F, Tumor DUSP6 mRNA expression was measured 3 hours after the last dose of SHP099 for each respective xenograft modeland normalized to its respective control. Data plotted are the mean percent fold change in DUSP6� SEM (n¼ 3). Each model is color coded with respect to itsKRAS genetic status as in E.






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Figure 4.

Efficacy of SHP2 loss or inhibition in KRAS-mutant tumors is tumor intrinsic. A, In vitro tube formation assay in HUVEC treated with 5 mmol/L SHP099. B, Tumorappearance of primary human colorectal xenograft model HCOX4087 at the end of treatment as described in Supplementary Fig. S3A. C, In vivomRNAexpression of mouse endothelial cell marker, CD31, in MIA PaCa-2 and T3M-4 xenografts evaluated after treatment with SHP099 (100mg/kg daily) or withselective VEGFR2 inhibitor BFH722 at 3 mg/kg daily from Supplementary Fig. S3B and S3C. Data plotted are percent fold change relative to the untreated control� SEM (n¼ 3). D, The growth rates of a SHP2 CRISPR knockout clone in MIA PaCa-2 cells (as demonstrated by immunoblot: P, parental line; WT, clone withoutSHP2 deletion; SHP2 crKO, SHP2 crispr knockout) in 2D and 3D, respectively. E, In vivo subcutaneous implantation of MIA PaCa-2 SHP2 crKO cells compared withthe parental control group. Data plotted are tumor volumemeans over time� SEM (n¼ 6). �� , P < 0.005. F, In vivo response in KRAS-mutant MIA PaCa-2 cellsto SHP099 (100mg/kg, daily) and to shRNA-induced depletion of SHP2. Doxycycline (Dox) and SHP099 treatment began at tumor randomization. Data plottedare tumor volumemeans over time� SEM (n¼ 6).G, Re-expression of Dox-inducible (TRE) SBP-SHP2T253M/Q257L mutant deficient in SHP099 binding in an SHP2knockout clone of MIA PaCa-2 cells and its 3D growth in the absence and presence of Dox or SHP099. DMSO or SHP099 (5 mmol/L) was added after 24 or48 hours of treatment with Dox at 0.1 mg/mL.

Hao et al.





KRAS-mutant cancers depend on upstream RTKsBecause SHP2 is a node downstream of RTKs, we hypothesized

that the effect of SHP2 inhibition in KRAS-mutant cancerscould be recapitulated with RTK inhibitors. We first evaluatedthe efficacy of an RTK inhibitor cocktail, targeting ERBBs, FGFRs,and MET, across a panel of KRAS-mutant cell lines in both 2Dand 3D culture conditions (Fig. 5A; Supplementary Table S4).Similar to treatment with SHP099, we found that the modestefficacy of the RTK inhibitor cocktail in 2D culture was alsoenhanced in the 3D growth conditions (Figs. 5A and 1E). Wenext confirmed these results in vitro and in vivo in both the MIAPaca-2 and KP4 cell lines (Fig. 5B–E). As observed with SHP099(Fig. 1C), efficacy of the RTK-inhibitor cocktail (Fig. 5B) in theMIA Paca-2 was improved in the 3D setting as evidenced bya shift in the antiproliferation IC50. In vivo efficacy of an EGFRmAb, cetuximab, or with an FGFR1-3 inhibitor, BGJ398, in theMIA PaCa-2 tumors resulted in modest tumor growth inhibition,64% T/C and 100% T/C, respectively. In contrast, combination ofthese two inhibitors resulted in a significant improvement inefficacy (26% T/C); however, SHP099 remained the most activetreatment in this xenograft model (7% T/C), presumably due tothe ability of SHP099 to inhibit signaling from additionalupstream RTKs, and potentially impacting other signaling path-ways (Fig. 5C).

Consistent with the antitumor efficacy, MAPK pathway sup-pression, as assessed by p-RSK3 levels, was reduced by SHP099 orthe combination of cetuximab and BGJ398, but not cetuximab orBGJ398 alone (Supplementary Fig. S4A). In contrast, p-ERK levelswere comparable in all treatment groups (SupplementaryFig. S4A) and only DUSP6 suppression was observed by SHP099(Supplementary Fig. S4A), likely due to its well-known tightregulation and the use of end of efficacy samples. Interestingly,there is a drastic p-SHP2 downregulation in MIA PaCa-2 xeno-grafts and 3Dorganoidswhen comparedwith the cells cultured in2D, consistent with the p-SHP2 reduction observed in 3D culturewhile p-AKT decrease in tumors is less pronounced comparedwihthe decrease in 3D spheroids (Supplementary Fig. S4B). Similarly,the p-EGFR and p-FRS2 signal in MIA PaCa-2 tumors were verylow, preventing us to understand the EGFR and FGFR activationstatus and responses to cetuximab and BGJ398 (SupplementaryFig. S4A). We also evaluated the KP4 model, a model that uponfurther analysis was determined to be dependent on MET due tooverexpression of the MET-ligand, HGF (SupplementaryFig. S4C). In KP4 we tested sensitivity in 3D and in vivo towardcapmatinib (Fig. 5D and E). Similar to the MIA PaCa-2 model,SHP099 treatment in the KP4 model trended toward betterefficacy than that of the MET inhibitor, although the results werenot statistically significant. Consistentwith the antitumor efficacy,p-RSK3 levels were modestly reduced by SHP099 or capmatinib(Supplementary Fig. S4D). Comparable with the MIA PaCa-2,p-SHP2, and p-MET levels, but not p-AKT levels, were also down-regulated in KP4 tumors and 3Dorganoids compared to KP4 cellscultured in 2D (Supplementary Fig. S4E). To further test ourhypothesis that in vivo sensitivity to SHP099 can potentially berelated to sensitivity to RTK inhibitors, we evaluated efficacy of anRTK inhibitor combination in the KRASQ61H-mutant T3M-4model. Combination of an FGFR1-3 inhibitor (BGJ398), METinhibitor (capmatinib), and EGFR inhibitor (cetuximab) waswell-tolerated and resulted in significant antitumor activity whencompared with the control and was comparable with that ofSHP099 (Fig. 5F). Given that comparable antitumor activity was

observed between SHP099 and RTK inhibitor treatment, combi-nation strategies with SHP2 inhibitors are warranted to deepenthe in vivo response. Taken together, these data demonstrate thatRTK activity is responsible for the activation of SHP2 in KRAS-mutant tumors and support further investigation toward the useof SHP2 inhibitors and combination partners to treat RTK-dependent KRAS-mutant cancer.

DiscussionIn this report, stimulated by the observation that a small subset

of KRAS-mutant cells showed sensitivity to the allosteric SHP2inhibitor SHP099, we profiled a panel of KRAS-mutant cell linesgrown asmulticellular spheroids and revealed an unexpected andwidespread sensitivity to SHP2 inhibition when KRAS-mutantcancer cells are grown in 3D. The efficacy of SHP099 in KRAS-mutantmodels was further confirmed in vivo and recapitulated bySHP2 depletion or knockdown in cancer cells, suggesting a tumorcell intrinsic mechanism (e.g., in the MIA PaCa-2 model bearingKRASG12C homozygous mutations). Significant MAPK pathwaysuppression by SHP099, albeit to a lesser extent compared withthe MEK inhibitor, trametinib, was observed in vivo, 3D, and 2Dgrowth conditions, in all models bearing KRAS G12 alterationstested and to some extent in KRAS G13 and Q61mutant models.However, MAPK suppression may not fully explain the activity ofSHP099 in all KRAS-mutant models and other SHP2-dependentbut MAPK-independent mechanismsmay contribute to the activ-ity of SHP099 in vivo, as illustrated in Supplementary Fig. S4F.

The enhanced activity of SHP099 in KRAS-mutant cells in 3Dcompared with 2D conditions, despite a similar degree of MAPKpathway suppression and rebound (Figs. 1C and 2A), is intrigu-ing. The altered sensitivity of cancer cells to anticancer therapies in3D growth conditions compared with 2D conditions has previ-ously been reported in KRAS-mutant cancer (22), HER2þ breastcancer (37), and colorectal cancer (35). However, the mechan-isms proposed to be responsible for these differential responses in2D and 3D are not consistent and are possibly due to lineage andtreatment specific differences. We observed a decreased baselinelevel of p-ERK and p-AKT and more importantly discovered adownregulation of SHP2 in three KRAS-mutant cell lines culturedin 3D conditions, manifested as either decreased SHP2 proteinlevels (transcriptional mechanism for MIA PaCa-2) or decreasedphosphorylation at the Y542 site. A similar p-SHP2 downregula-tionwas observed inMIA PaCa-2 and KP4 tumors compared with2D growth conditions, as well as p-MET, but not p-AKT (Sup-plementary Fig. S4B and S4E). Those signaling differences in3D and in vivomay partially explain why the sensitivity to SHP2inhibitors of KRAS mutant cells is particularly revealed under3D and in vivo conditions. Although one may conclude that the3D response is more predictive of in vivo efficacy, the T3M-4model that showed a response to SHP099 in vivo did not exhibitsensitivity in 3D. It is possible that the role of SHP2 in KRAS-mutant signaling is dependent on a complex tumor microen-vironment beyond what a tumor spheroid in vitro model canreveal. It is also possible that different mechanisms couldaccount for the 3D sensitivity and in vivo sensitivity to SHP099in different KRAS-mutant models, as the p-AKT levels in 2Dmore resemble that in xenograft tumors of MIA PaCa-2 andKP4 (Fig. 2A; Supplementary Fig. S4B and S4E).

KRAS andMAPK pathway activation are regulated by upstreamsignaling proteins, such as EGFR, FGFR, MET (48–51), as well as






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KRAS-mutant cancers depend on upstream RTK signaling.A, Effect of an RTK inhibitor cocktail consisting of lapatinib, BGJ398, and capmatinib in a panel ofKRAS-mutant cell lines cultured in 2D and 3D. KRASmutation of each cell line is indicated by the symbol shapes, and response data are located in SupplementaryTable S4. B, Effect of an RTK inhibitor cocktail consisting of lapatinib, BGJ398, and capmatinib (10 mmol/L for each in the highest concentration) on proliferationof MIA PaCa-2 cells for 6 days in 2D and 3D culture, respectively. Blue and green dotted lines are the percentage of day 0 reading of DMSO-treated cellsnormalized to that of day 6 in 2D and 3D, respectively. The black hyphenated lines visualize IC50 values that are reported in the graph. Data are representative oftwo independent experiments each performed in triplicates. Error bars, SEM. C, In vivo efficacy of EGFR inhibitor, cetuximab (20mg/kg, 2�weekly), FGFR1-3inhibitor, BGJ398 (15 mg/kg, daily), combination of cetuximab and BGJ398 and SHP099 (100mg/kg, daily) in MIA PaCa-2 cells implanted subcutaneously. Dataplotted are tumor volumemeans� SEM (n¼ 8). �� , P < 0.01; �� , P < 0.005. D, Effect of MET inhibitor capmatinib on the proliferation of KP4 cells for 6 days in 2Dculture and 3D culture, respectively. E, In vivo efficacy of MET inhibitor, capmatinib (10 mg/kg, daily), and SHP099 (100mg/kg, daily) in KP4 cells implantedsubcutaneously. Data plotted are tumor volumemeans� SEM (n¼ 7). �� , P < 0.01. F, In vivo efficacy of SHP099 (100mg/kg, daily) and combination of BGJ398(15 mg/kg, daily), capmatinib (10 mg/kg, daily), and cetuximab (20mg/kg, twice a week) in T3M-4 cells implanted subcutaneously. Data plotted are tumorvolumemeans� SEM (n¼ 8). �� , P < 0.005. NS, nonsignificant.

Hao et al.





by the SHP2 protein tyrosine phosphatase and the RAS-GEF,SOS1 (7, 17). Indeed, tyrosine kinase inhibitors have been shownto provide overall survival benefit in patients (50, 52, 53); how-ever, retrospective analysis has also demonstrated that the activityof these selective inhibitors is mostly restricted to tumors har-boring wild-type RAS or RAF (54–57). Furthermore, in vitro 2Dscreens of RTK and SHP2-inhibitors in cell lines confirmed thattheir activity was dependent on the wild-type KRAS/BRAF geneticstatus (Fig. 1A and B; ref. 15). These data had long supported thenotion that gain-of-function KRAS mutations, commonly of theG12, G13, Q61 form, were constitutively active and that targetingupstream signaling would be ineffective. However, this paradigmhas shifted in recent years, with several publications demonstrat-ing biochemically that the KRASG12Cmutation is not locked in theconstitutively active form, but maintains intrinsic GTP-hydrolyticactivity (19–21). In fact, these data helped the development of aselective and active KRASG12C inhibitor, ARS853 (20). Togetherwith the newly appreciated KRAS biology and the effects ofSHP099 in KRAS-mutant cancer, we demonstrate that KRAS-mutant cancers are still dependent on upstream receptor tyrosinekinase signaling, although the specific RTK's that regulate theSHP2-KRAS signaling axis appear to be cancer cell-line specific(Fig. 5). Furthermore, the activity observed with SHP099 appearsto be comparable to that of RTK-inhibition in these celllines, where only tumor stasis is observed. It is possible thattreatment of cancers with a SHP2 inhibitor alone could preventpathway feedback reactivation of the MAPK pathway or blockRTK-bypass mechanisms where upregulation of other RTK'sunder treatment may contribute to tumor resistance (33). How-ever, to induce tumors into regression the identification of opti-mal clinical combination agents with a SHP2 inhibitor warrantsfurther investigation.

With the generation of the first selective SHP2 inhibitor,SHP099 (15, 16), we demonstrated that SHP099 was potentlyactive in RTK-driven cancers harboring wild-type KRAS andBRAF (15). Consistent with the notion that KRAS-mutant cancerswould be insensitive to SHP2 inhibition, we showed limitedactivity of SHP099 in KRAS-mutant cancer cells grown in 2Dculture. However, broad evaluation of SHP099 in KRAS-mutantcell lines cultured as either tumor spheroids or as in vivo xenograftmodels revealed significant antitumor activity (Figs. 1 and 3).These findings mirror that which has been recently and indepen-dently reported, that KRAS-mutant cancers, specifically KRASG12C

and KRASG12D variants still depend on SHP2 activity (17, 22, 23).Here, we show that the in vivo activity of SHP099 in theMIAPaCa-2 KRASG12C model in an immunocompromised mouse setting isdue to tumor cell-intrinsic mechanisms. However, this does notexclude the potential for additional antitumor activity due tonontumor cell-intrinsic mechanisms that a SHP2-inhibitor mayimpart in humans, where SHP2-mediated effects may includeboth tumor cell autonomous effects and effects on stromal cellsand immune cells (25, 58). In T-cells, SHP2 was shown to berecruited by programmed cell death-1 (PD1) to dephosphorylateand inactivate costimulatory receptor CD28, which suppresses T-cell activation (25). SHP099, intraperitoneally dosed, has signif-icant anti-tumor efficacy in the CT-26 syngeneic model grown inimmunocompetent mice, but little efficacy when the model isgrown in immunodeficient nude mice or in vitro, although thedose and route of administration of SHP099 that were used arequestionable (59). Using myelomonocytic cell–specific Shp2knockout mice, Shp2 ablation in myeloid cells was shown to

inhibit melanoma growth by potentiating macrophage produc-tion of T-cell chemoattractant CXCL9 in response to IFN-g andtumor cell-derived cytokines, thereby facilitating the tumor infil-tration of IFNg-producing T cells (60). In addition, SHP2 inhi-bition synergized with PD-1 blockade in the MC-38 murinesyngeneic colon cancer model (59).

The dependence of multiple RTKs/SHP2 for activation of themutant KRAS signaling and the potential immunomodulatoryeffects of SHP2 inhibition provides a strong clinical rationale forthe utility of a SHP2 inhibitor for treating KRAS-mutant cancers,where treatment options are currently limited.

Disclosure of Potential Conflicts of InterestS. Kovats is the scientific technical leader at Novartis Institute for Biomedical

Research. M. Shirley is an investigator at Novartis Institute for BiomedicalResearch. M.J. Meyer is a former employee of Novartis Institute of BiomedicalResearch; is the Head, Discovery Pharmacology and In Vivo Biology, at BMS;and has ownership interest (including patents) in Novartis and BMS. M.J.LaMarche is a senior investigator at Novartis Institutes of Biomedical Researchand has ownership interest (including patents) in NVS. S. Moody is the clinicalprogram leader at Novartis Institutes for Biomedical Research. S.J. Silver is aformer employee and stockholder at Novartis Institutes for BiomedicalResearch.D.D. Stuart is an executive director atNovartis Institutes of BiomedicalResearch and has ownership interest (including patents) in Novartis. T.J.Abrams is a senior investigator at Novartis Institutes of Biomedical Researchand has ownership interest (including patents) in Novartis. J. Williams is anexecutive director at Novartis, has a commercial research grant for Novartis, andhas ownership interest (including patents) in Novartis. J.A. Engelman is theglobal oncology head at Novartis Institutes of Biomedical Research and hasownership interest (including patents) in Novartis. M. Mohseni is an investi-gator at Novartis Institutes of Biomedical Research and has ownership interest(including patents) inNovartis. No potential conflicts of interest were disclosedby the authors.

Authors' ContributionsConception and design: H.-X. Hao, H. Wang, C. Liu, S. Kovats, M.J. Meyer,M.J. LaMarche, G. Caponigro, T.J. Abrams, J.A. Engelman, M. MohseniDevelopment of methodology: H. Wang, C. Liu, S. Kovats, B. Pant, J. Lim,M. Fleming, S.J. Silver, T.J. Abrams, S. GoldoniAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C. Liu, S. Kovats, R. Velazquez, H. Lu, B. Pant,M.J. Meyer, M. Pu, J. Lim, M. Fleming, L. Alexander, A. Farsidjani,M.J. LaMarche, J. WilliamsAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):H.-X. Hao, H. Wang, C. Liu, S. Kovats, H. Lu, B. Pant,M. Shirley, M.J. Meyer, M. Pu, J. Lim, M. Fleming, A. Farsidjani, M.J. LaMarche,S.J. Silver, D.D. Stuart, S. Goldoni, M. MohseniWriting, review, and/or revision of the manuscript: H.-X. Hao,C. Liu, R. Velazquez, B. Pant, M.J. Meyer, S. Moody, G. Caponigro,P.S. Hammerman, J. Williams, S. Goldoni, M. MohseniAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H. Wang, B. Pant, M. Pu, A. Farsidjani,M. MohseniStudy supervision: H.-X. Hao, M.J. LaMarche, S.J. Silver, G. Caponigro,D.D. Stuart, P.S. Hammerman, J.A. Engelman, M. Mohseni

AcknowledgmentsWe thank the following Novartis colleagues, Yingnan Chen, Brant Firestone,

Ho Man Chan, Michael Acker, Hui Gao, Alice Loo, Suzanna Clark, and LisaDuong for their helpful technical advice, discussion of data interpretation, andsuggestions of experiment design on the manuscript.

The costs of publicationof this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 15, 2019; revised July 10, 2019; accepted August 16, 2019;published first August 22, 2019.






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Published OnlineFirst August 22, 2019.Mol Cancer Ther Huai-Xiang Hao, Hongyun Wang, Chen Liu, et al. KRAS-Mutant CancersTumor Intrinsic Efficacy by SHP2 and RTK Inhibitors in

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