Allosteric SHP2 Inhibitor, IACS-13909, Overcomes EGFR ......CANCER RESEARCH | TRANSLATIONAL SCIENCE Allosteric SHP2 Inhibitor, IACS-13909, Overcomes EGFR-Dependent and EGFR-Independent

CANCER RESEARCH | TRANSLATIONAL SCIENCE

Allosteric SHP2 Inhibitor, IACS-13909, OvercomesEGFR-Dependent and EGFR-Independent ResistanceMechanisms toward Osimertinib A C

Yuting Sun1, Brooke A. Meyers1, Barbara Czako2, Paul Leonard2, Faika Mseeh2, Angela L. Harris1, Qi Wu2,Sarah Johnson1, Connor A. Parker2, Jason B. Cross2, Maria Emilia Di Francesco2, Benjamin J. Bivona1,Christopher A. Bristow1, Jason P. Burke2, Caroline C. Carrillo1, Christopher L. Carroll2, Qing Chang1,Ningping Feng1, Guang Gao1, Sonal Gera1, Virginia Giuliani1, Justin K. Huang1, Yongying Jiang2,Zhijun Kang2, Jeffrey J. Kovacs1, Chiu-Yi Liu1, Anastasia M. Lopez1, Xiaoyan Ma1, Pijus K. Mandal2,Timothy McAfoos2, Meredith A. Miller1, Robert A. Mullinax1, Michael Peoples1, Vandhana Ramamoorthy1,Sahil Seth1, Nakia D. Spencer1, Erika Suzuki1, Christopher C. Williams2, Simon S. Yu2, Andy M. Zuniga1,Giulio F. Draetta3, Joseph R. Marszalek1, Timothy P. Heffernan1, Nancy E. Kohl4, and Philip Jones2

ABSTRACT◥

Src homology 2 domain-containing phosphatase (SHP2) is aphosphatase that mediates signaling downstream ofmultiple recep-tor tyrosine kinases (RTK) and is required for full activation of theMAPK pathway. SHP2 inhibition has demonstrated tumor growthinhibition in RTK-activated cancers in preclinical studies. The long-term effectiveness of tyrosine kinase inhibitors such as the EGFRinhibitor (EGFRi), osimertinib, in non–small cell lung cancer(NSCLC) is limited by acquired resistance. Multiple clinicallyidentified mechanisms underlie resistance to osimertinib, includingmutations in EGFR that preclude drug binding as well as EGFR-independent activation of the MAPK pathway through alternateRTK (RTK-bypass). It has also been noted that frequently a tumorfrom a single patient harbors more than one resistance mechanism,and the plasticity between multiple resistance mechanisms couldrestrict the effectiveness of therapies targeting a single node of theoncogenic signaling network. Here, we report the discovery of

IACS-13909, a specific and potent allosteric inhibitor of SHP2, thatsuppresses signaling through the MAPK pathway. IACS-13909potently impeded proliferation of tumors harboring a broad spec-trum of activated RTKs as the oncogenic driver. In EGFR-mutantosimertinib-resistant NSCLC models with EGFR-dependent andEGFR-independent resistance mechanisms, IACS-13909, adminis-tered as a single agent or in combination with osimertinib, potentlysuppressed tumor cell proliferation in vitro and caused tumorregression in vivo. Together, our findings provide preclinical evi-dence for using a SHP2 inhibitor as a therapeutic strategy inacquired EGFRi-resistant NSCLC.

Significance: These findings highlight the discovery of IACS-13909 as a potent, selective inhibitor of SHP2 with drug-likeproperties, and targeting SHP2 may serve as a therapeutic strategyto overcome tumor resistance to osimertinib.

IntroductionThe Src homology 2 domain-containing phosphatase 2 (SHP2,

encoded by PTPN11) is a critical regulator of oncogenic MAPKsignaling. The SHP2 protein contains N-terminal and C-terminalSrc homology 2 (SH2) domains, a protein tyrosine phosphatase(PTP) domain, and a C-terminal tail. The inactive SHP2 protein ismaintained in a closed state by interdomain interactions betweenthe N-terminal SH2 and PTP domains, preventing substrate access

to the active site. SHP2 mutations at the interface of the SH2 andPTP domains, such as the somatic mutations found in juvenilemyelomonocytic leukemia and the germline mutations found inNoonan Syndrome, open and activate the protein (1, 2).

In addition to being an oncogenic driver in raremalignancies, SHP2critically mediates MAPK pathway signaling downstream of a broadspectrum of receptor tyrosine kinases (RTK), including EGFR, HER2,MET, and PDGFR (3–7). The intracellular phospho-tyrosine residueson activated RTKs interact with the SH2 domains on wild-type (WT)SHP2, resulting in an open, active conformation of SHP2, andsubsequent activation of the downstream MAPK signaling cascade.SHP2 also sits upstream of RAS in the MAPK pathway and fullactivation of RAS requires input from SHP2, particularly the nucle-otide cycling mutant of RAS (i.e., G12C; refs. 8–11).

The development of multiple generations of tyrosine kinase inhi-bitors (TKI) has transformed the clinical landscape of non–small celllung cancer (NSCLC), yet acquired resistance remains as a majorchallenge. Osimertinib is a mutant-selective, third-generation EGFRinhibitor (EGFRi) that targets both EGFR-activating mutations (e.g.,exon 19 deletion and L858R) and EGFR-dependent on-target resis-tance mutation toward the first-generation EGFRi (i.e., T790M;ref. 12). It is currently a first-line therapy for EGFR-mutant (EGFRmut)NSCLC, with average progression-free survival of approximately19 months in previously untreated patients (13). Clinical and

1TRACTION - Translational Research to AdvanCe Therapeutics and Innovation inONcology, TheUniversity of TexasMDAnderson Cancer Center, Houston, Texas.2Institute for Applied Cancer Science (IACS), The University of Texas MDAnderson Cancer Center, Houston, Texas. 3Department of Genomic Medicine,The University of Texas MD Anderson Cancer Center, Houston, Texas. 4NavirePharma, San Francisco, California.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Yuting Sun, The University of Texas MD AndersonCancer Center, 1881 East Rd, Houston, TX 77054. Phone: 713-794-4298; Fax: 713-792-6882; E-mail: [email protected]

Cancer Res 2020;80:4840–53

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preclinical studies have revealed numerous resistance mechanisms.Among these, EGFR-dependent mechanisms such as resistance muta-tions in EGFR (e.g., C797S and reversal to WT EGFR) occur in 20%–50% of relapsed patients. Other clinically observed resistance mechan-isms, which also apply to earlier generations of EGFRi, includeactivation of alternate RTKs (e.g., MET, FGFR, HER2, and IGF1R),PIK3CA mutations, and mutations in RAS/RAF pathway that main-tain downstream ERK activation (14–16). In addition, nonsignalingmechanisms such as epithelial–mesenchymal transition (EMT), acqui-sition of stem-like properties, and metabolic rewiring have also beenreported in preclinical models (17, 18). Importantly, it has been notedthat tumor from a single patient may harbor more than one resistancemechanisms (16, 19), suggesting that with oncogenic shock fromEGFRi, the tumor may switch to multiple alternate drivers. Forexample, EGFR C797S mutation and MET amplification have beenreported to coexist in the same tumor sample from a patient whorelapsed on osimertinib treatment (16). The heterogeneity and plas-ticity in resistance mechanisms make it challenging to treat patientswith a therapy targeting a single RTK.

Because SHP2 critically mediates the signaling of multipleRTKs, and several resistance mechanisms toward osimertinib arethrough RTK signaling, we hypothesize that a SHP2 inhibitor maybe effective in addressing the heterogeneous mechanisms of osi-mertinib resistance. In this study, we report the discovery of IACS-13909, a novel, potent, and selective allosteric inhibitor of SHP2,that suppresses signaling through the MAPK pathway and inhibitsproliferation of RTK-activated tumors in vitro and in vivo. Impor-tantly, we provide preclinical data showing IACS-13909, eitheradministered as a single agent or in combination with osimertinib,potently suppresses tumor cell proliferation in vitro and causestumor regression in vivo in tumors with EGFR-dependent andEGFR-independent resistance mechanisms.

Materials and MethodsAdditional/detailed methods are provided in Supplementary Data.

In vitro enzymatic assayPhosphatase activity of full-length SHP2 or SHP2 phosphatase

domain was measured using fluorogenic 6,8-difluoro-4-methylum-belliferyl phosphate (Molecular Probes) as the substrate. Detailedmethod is described Supplementary Data.

X-ray crystallographyCocrystals of SHP2:IACS-13909 were generated and a 2.4-Å struc-

turewas solved byX-ray crystallography.Details for crystal generation,structure determination, and data analysis are provided in Supple-mentary Data.

Cell culture and generation of engineered linesAll cell lines, unless specified, were obtained from an internal cell

bank, which conducted short tandem repeat (STR) finger printing andPCR-based Mycoplasma testing on all cryopreserved batches. STRfinger printing was also conducted with all engineered cell lines andderivatives. Unless specified, experiments were conducted with cells<6 weeks after thawing.

All cells were cultured at 37�C with 5% CO2. KYSE-520, NCI-H1975, NCI-H1975 CS, LS411N, HCC827, and HCC4006 cells werecultured in RPMI1640 Medium (Thermo Fisher Scientific) supple-mented with 10% FBS (Sigma). MIA PaCa-2, MV-4-11, and 293T cellswere cultured in high glucose DMEM (Thermo Fisher Scientific)

supplemented 10% FBS. MV-4-11-Luc cells were from the Experi-mental Therapeutics Core at Dana-Farber Cancer Institute (Boston,MA) and cultured in high-glucose DMEM supplemented with 10%FBS and 1 mg/mL puromycin.

The HCC827-ER1 cells that harbor MET amplification (CrownBioscience UK) were cultured in RPMI1640 medium supplementedwith 10% FBS and 42 mmol/L erlotinib (20). The cell line was derivedfrom HCC827 (ATCC), at Crown Bioscience, by culturing the cells inthe presence of escalated concentrations of erlotinib. The HCC4006-OsiR cells were generated by culturing HCC4006 cells in the presenceof 1 mmol/L osimertinib for approximately 3 months, and weremaintained in RPMI1640 medium supplemented with 10% FBS and1 mmol/L osimertinib.

NCI-H1975 CS cells that harbor EGFRL858R/T790M/C797S mutationwere generated through CRISPR Cas9–mediated point mutation atEGFR C797 site, by Synthego Corporation. To generate these cells,ribonucleoproteins containing the Cas9 protein and synthetic, chem-ically modified single guide RNA were electroporated into the cellsusing Synthego's optimized protocol. Editing efficiency was assessedupon recovery, 48 hours after electroporation. Genomic DNA wasextracted from a portion of the cells, PCR amplified, and sequencedusing Sanger sequencing. The resulting chromatograms were pro-cessed using Synthego Inference of CRISPR edits software (ice.synthego.com).

KYSE-520 cells stably overexpressing SHP2 WT or SHP2 P491Qwere generated by infecting the parental cells with concentratedlentiviruses, and cultured in the media of the parental cells with1 mg/mL Puromycin (Thermo Fisher Scientific). The cells were splitwhenever needed, cultured for approximately 2 weeks, and frozendown for future experiments.

Cell proliferation assaysIn vitro clonogenic assayswere conductedwith adherent lines plated

in 12-well or 24-well plates, treated for 2weeks, and stainedwith crystalviolet. Ex vivo spheroid proliferation assay was conducted with cellsfreshly isolated from patient-derived xenograft (PDX), plated in U-bottom ultra-low attachment 96-well plates (Corning) without matrix,and treated for 6 days. Detailed procedures are described in Supple-mentary Data.

RNA-sequencing (QuantSeq) and data analysisRNA libraries were prepared with the QuantSeq 30 mRNA-Seq

FWDKit (Lexogen), following the manufacturer's standard protocols.Briefly, libraries were generated with 500 ng total RNA as input and 11cycles of PCR amplification of the cDNA. Batches of up to 40 sampleswere multiplexed and each batch was run on NextSeq 500 (Illumina)using the High Output Kit v2 (Illumina).

Sample analyses were conducted using R Bioconductor. Transcriptcompatibility counts were obtained with kallisto (v0.44.0; ref. 21)running the pseudomode with GENCODE 23 transcript annota-tions (22). Gene counts were obtained by summing all reads thatuniquely mapped, and differential expression analysis was carried outusing DESeq20s (23) default settings. Heatmaps were generated inGraphPad Prism 8.0.

Mouse studiesAll in vivo work was either approved by the Institutional Animal

Care and Use Committee of MD Anderson Cancer Center (Houston,TX) or by the relevant committee of the testing facility. Female micewere used, and body weight was typically 20–28 g when treatmentwas started.

Targeting SHP2 to Overcome Osimertinib Resistance

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All subcutaneousmodels were implanted with 50%Matrigel (Corn-ing). Cell numbers andmouse strains were: KYSE-520, 3� 106 inNSGmice (The Jackson Laboratory); NCI-H1975, 1 � 106 in CD-1 Nudemice (Charles River Laboratories); and NCI-H1975 CS, 3 � 106 inNSG; and HCC827 and HCC827-ER1, 5 � 106 in athymic nude mice(Envigo). Tumor size wasmeasured with caliper and calculated using astandard formula: length� width2/2. Dosing volume was 10 mL/kg/day. IACS-13909 was formulated in 0.5% methylcellulose, andosimertinib/erlotinib in 0.5% HPMC. For the combination studies,IACS-13909 was dosed in the morning and osimertinib was dosedin the afternoon, with a 6-hour interval in between. To pool tumormeasurements from independent experiments, biweekly measure-ments differing by 1 day across studies were considered as at thesame timepoint.

For studies with the MV-4-11 orthotopic model (ExperimentalTherapeutics Core, Dana-Farber Cancer Institute, Boston, MA), NSGmice were implanted with 2 � 106 MV-4-11-Luc cells (250 mL)intravenously. Mice were enrolled into treatment groups using totalflux bioluminescence value, 2 days postimplantation. After dosingended, all animals were monitored for survival, and euthanized oncemorbidity and/or stage III paralysis was observed.

Data plotting and statistical analysisUnless specified, data plotting and statistical analysis were con-

ducted using GraphPad Prism 8.0. Graph with error bars representmean � SEM.

ResultsIACS-13909 is a potent and selective allosteric inhibitor ofSHP2

To discover novel SHP2 inhibitors with drug-like properties, weutilized structure-based design principles starting from known SHP2allosteric inhibitors, and identified IACS-13909 (Fig. 1A). In anin vitro enzymatic assay, IACS-13909 potently suppressed the phos-phatase activity of purified full-length, recombinant human SHP2protein with an IC50 of approximately 15.7 nmol/L (Fig. 1B). Incomparison, in a similar assay using the SHP2 phosphatase domain,IACS-13909 did not suppress phosphatase activity at concentrationsup to 50,000 nmol/L, the highest concentration tested (Fig. 1C),suggesting IACS-13909 acts outside the phosphatase domain. The Kd

of IACS-13909 binding to SHP2 was approximately 32 nmol/L, asdetermined by isothermal titration calorimetry analysis (Supplemen-tary Fig. S1A). IACS-13909 is highly selective for SHP2.When tested at10 mmol/L against a panel of 22 phosphatases, the compound onlyshowed significant inhibition of SHP2 (>50% inhibition; Supplemen-tary Table S1). It is notable that IACS-13909 demonstrated noinhibition of full-length SHP1, the phosphatase that is structurallymost similar to SHP2.

To elucidate where IACS-13909 interacts with SHP2 protein, wesolved the crystal structure of SHP21–530 with IACS-13909 at 2.4 Åresolution with Rfree of 0.270 (PDB¼ 6WU8; Fig. 1D; SupplementaryTable S2). The refined structure contained two protomer chains ofSHP21–530 and two molecules of IACS-13909 in the asymmetric unit.The crystal structure confirmed that the compound bound outside theactive site, at the interface between the phosphatase domain (gray) andthe C-terminal SH2 domain (cyan), a key allosteric pocket of theprotein (24), and stabilizes the inactive state of the enzyme. Keyhydrogen bond interactions were observed between the backbonecarbonyls of Glu110 and Phe113 and the basic amine group ofIACS-13909, as well as between the backbone carbonyl of Glu250

and the pyrazole N–H of the compound. A water molecule bridgesbetween the sidechains of Thr219 and Arg111 and the pyrazine core ofthe compound and we observed cation-

Qstacking interactions

between the Arg111 sidechain and the dichlorobenzene of IACS-13909. Together, these data confirm that IACS-13909 is a directallosteric inhibitor of SHP2.

IACS-13909 inhibits the proliferation and MAPK pathwaysignaling of tumor cell lines driven by a broad spectrum of RTKsin vitro

Because SHP2 is a critical mediator of oncogenic signaling, a SHP2inhibitor might be useful as an anticancer agent (2). We evaluated thein vitro antiproliferative effect of IACS-13909 in a panel of 283 cancercell lines with diverse genomic drivers, using a 10-day two-dimensional proliferation assay. Among the exceptional responderlines (with GI50 ≤ 1 mmol/L), many harbored genetic alterations ofRTK or were RTK addicted (sensitive to TKI or RTK short hairpinRNA according to DRIVE; ref. 25). Particularly, all six cell lines withGI50 ≤ 100 nmol/L harbored RTK alterations, DK-MG (EGFR vIIIþ),BV-173 (BCR-ABL), KG-1 (OP2-FGFR1), KU812 (BCR-ABL), SW-13(ERBB4-IKZF2; ref. 26), and MV-4-11 (FLT3-ITD; SupplementaryFig. S1B). In addition, BRAFV600mutation appeared to be a predictorof IACS-13909 resistance, with 19 of 23 BRAFV600–mutated cell lineshaving GI50 > 5 mmol/L. Consistent with the proliferation data, IACS-13909 suppressed pERK in RTK-dependent lines, such as KYSE-520(EGFRamp; Supplementary Fig. S1C) and MV-4-11 (FLT3-ITD; Sup-plementary Fig. S1D), but did not suppress pERK or pMEK in LS411Ncells harboring BRAFV600E (Supplementary Fig. S1E). It is note-worthy that majority of KRASmut cell lines in this analysis wereresistant to IACS-13909 (Supplementary Fig. S1B), likely due to lowcoverage of cell lines expressing the nucleotide-cycling KRASG12C

mutant in this panel and limitations of the two-dimensional culturesystem in evaluating KRASmut cancers. Together, these data dem-onstrate the antitumor activity of IACS-13909 in cancer cell linesharboring a broad spectrum of activated RTKs, consistent withliterature (24).

IACS-13909 inhibits the proliferation and MAPK pathwaysignaling in RTK-activated cancer cells in vitro due to on-targetSHP2 inhibition

To ensure that the antiproliferative effect of IACS-13909 in RTK-activated cancer cell lines in vitro was due to inhibition of SHP2, weleveraged the SHP2 proline 491 to glutamine (P491Q) mutant. On thebasis of the X-ray crystal structure, Pro491 lines the allosteric bindingpocket of SHP2 adjacent to the pyrazolopyrazine ring of IACS-13909.Sequence and structural alignment with SHP1 (PDB¼ 3PS5) showedglutamine 485 in this position in SHP1 and suggested that a P491Qmutation will abolish IACS-13909 binding to SHP2 due to stericclashes with the glutamine side chain (Fig. 1D), but should still yielda catalytically competent protein (8). Therefore, we stably overex-pressed dsRed (control), SHP2WT, or SHP2 P491Q in the KYSE-520cells, an EGFRamp esophageal cancer cell line.Western blotting showedthat exogenous SHP2 was expressed at a much higher level thanendogenous SHP2 (Fig. 1E). In control cells or cells overexpressingSHP2 WT, IACS-13909 potently suppressed pERK and pMEK levels,but not in cells overexpressing SHP2 P491Q. Similarly, in an in vitroclonogenic assay, whereas IACS-13909 potently suppressed the pro-liferation of control cells and cells overexpressing SHP2 WT (GI50 <1mmol/L), overexpression of SHP2 P491Q significantly reduced sensi-tivity of IACS-13909 (with 7.8-fold shift in IACS-13909 GI50; Fig. 1F).The rescue was specific to SHP2 inhibitor, because EGFRi, erlotinib,

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demonstrated identical sensitivity in control, SHP2 WT, and SHP2P491Q-overexpressing cells (Supplementary Fig. S1F and S1G). It isnoteworthy that SHP2 P491Q overexpression did not confer completeresistance of KYSE-520 cells to IACS-13909; this is likely because of thepresence of endogenous SHP2 in the mutant overexpressing KYSE-520 cells that confers some signaling through the MAPK pathway.Together, these data suggest that IACS-13909 suppresses cell prolif-eration and signaling through the MAPK pathway in RTK-dependenttumor cells due to its inhibitory effect on SHP2.

In vivo antitumor activity of IACS-13909 in models driven by abroad spectrum of RTKs

To determine the activity of IACS-13909 in vivo, we first evaluatedthe pharmacokinetic properties of IACS-13909 inmice, rats, and dogs.IACS-13909 demonstrated >70% bioavailability, low clearance rate,and half-lives of≥7hours across species, suggesting that the compoundis suitable for once per day oral dosing (Supplementary Table S3).

We selected twoRTK-dependent cell lines for in vivo evaluation, theEGFRamp esophageal cancer cell line, KYSE-520, as a representativesolid tumor cell line, and the FLT3-ITDþ acute myeloid leukemia cellline, MV-4-11, as a representative blood cancer cell line. In mice withestablished subcutaneous KYSE-520 tumors, IACS-13909 dosed orallyat 70 mg/kg once per day potently suppressed tumor growth, with100% tumor growth inhibition (TGI) observed following 21 days ofdosing (Fig. 2A). Importantly, the treatment was well-tolerated, withbodyweightmaintained throughout the study (Fig. 2B). A higher doseof IACS-13909, such as 100 mg/kg once per day, was not tolerated inmice, suggesting 70 mg/kg once per day is approximately the MTD ofIACS-13909 in mice.

To confirm that the in vivo antitumor efficacy of IACS-13909 wasdue to SHP2 inhibition, we analyzed KYSE-520 tumors and bloodfrom mice treated with different dosing levels of IACS-13909. ThemRNA levels of DUSP6, an ERK-dependent gene, were used as areadout of SHP2 activity andMAPKpathway signaling in tumors (27).IACS-13909 achieved dose-dependent plasma exposure at 24 hoursafter a single-dose treatment, and demonstrated dose-dependentsuppression of DUSP6 transcript levels in KYSE-520 tumors(Fig. 2C). An inverse correlation between tumor DUSP6mRNA leveland plasma concentration was observed. Among the doses tested,IACS-13909 at 60 or 80 mg/kg span the dose that resulted in tumorstasis in this model (Fig. 2A), maintainingDUSP6mRNA suppressionat >50% throughout the 24-hour dosing interval. These data demon-strate that IACS-13909 potently suppresses MAPK pathway signalingand inhibits growth of an RTK-dependent subcutaneous solid tumormodel in vivo.

We further tested the in vivo antitumor efficacy of IACS-13909 inthe FLT3-ITDþ MV-4-11 leukemia orthotopic model. Mice wereimplanted with MV-4-11 cells expressing luciferase through tail veininjection, and systemic tumor growth was rapidly established. Micewere randomized on the basis of tumor luminescence levels, and thentreated with IACS-13909 at different dosing levels for 5 weeks. Dose-dependent suppression of systemic tumor burden was observed(Fig. 2D and E), with IACS-13909 at 75 mg/kg once per day causingnearly 100% TGI. Importantly, consistent with the suppression oftumor burden, IACS-13909 extended the overall survival of themice ina dose-dependent manner (Fig. 2F). These data demonstrate the dose-dependent antitumor efficacy of IACS-13909 in an RTK-dependentdisseminated leukemia model.

Figure 1.

IACS-13909 is a potent and selective allosteric inhib-itor of SHP2. A, The structure of IACS-13909. B,Doseresponse of IACS-13909 in an in vitro enzymaticassay with purified full-length human SHP2, in thepresence of 1 mmol/L bistyrosylphorphorylated pep-tide. The dose–response curve is from a single rep-resentative experiment. The IC50was calculated from59 independent tests. C, The effect of IACS-13909 inan in vitro enzymatic assay with purified, humanSHP2 phosphatase domain. N ¼ 10. Because 50%inhibition was not achieved, IC50 was defined asabove the top tested concentration. D, Crystal struc-ture of IACS-13909 with purified human SHP2 pro-tein, at 2.4 Å, determined by X-ray crystallography.PDB¼ 6WU8. The phosphatase domain is highlight-ed in gray, C-SH2 in cyan, and N-SH2 in green. E, Theimpact of IACS-13909 on pERKT202/Y204 and pMEK1/2S217/221 levels in KYSE-520 cells overexpressingdsRED (control), SHP2 WT, or SHP2 P491Q mutant.Cells were treated with IACS-13909 for 2 hours andprocessed for Western blotting. F, The in vitro anti-proliferative effect of IACS-13909 on cells used in Edetermined by a 14-day clonogenic assay.N¼ 2 fromthe same experiment. This experiment was repeatedwith another allosteric SHP2 inhibitor with similarobservation.






IACS-13909 demonstrates antitumor activity in tumorsharboring EGFR-dependent resistance mutation in vitro andin vivo

Multiple EGFR TKIs (e.g., erlotinib, gefitinib, and osimertinib) arecurrently approved in the United States for the first-line treatment ofpatients with EGFR-activated metastatic NSCLC (13). Most patientson EGFRi treatment will ultimately experience disease progression,with acquired resistance being a major clinical challenge. EGFRmutations in the proximity of the compound binding site (e.g.,T790M for erlotinib/gefitinib and C797S or L792H for osimertinib)that preclude drug binding are clinically observed resistance mechan-

isms (14, 15). Considering that tumors with an EGFR resistancemutation still depend on EGFR and also that SHP2 is a criticalmediator of EGFR signaling, we hypothesized that IACS-13909 mighthave activities in these tumors.

To evaluate the effect of SHP2 inhibition on cancer cells harboringEGFR resistance mutations, we used NSCLC NCI-H1975 cells thatharbor both an EGFR-activating mutation (L858R) and resistancemutation (T790M). The NCI-H1975 cells are resistant to erlotinib andsensitive to osimertinib (12). In addition, we generated theNCI-H1975CS cells, in which EGFR C797S mutation was introduced through theCRISPR Cas9 technology (Supplementary Fig. S2). The NCI-H1975

Figure 2.

IACS-13909 suppresses proliferation andMAPKpathway signaling of RTK-activated tumors in vivo.A andB, Tumor growth curve (A) andmouse bodyweight change(B) of the KYSE-520 subcutaneous xenograft model in mice when treated with either vehicle (0.5%methylcellulose) or IACS-13909 at 70 mg/kg once per day (QD)orally (p.o.) for 21 days.N¼ 9mice per group.C, Plasma concentration of IACS-13909 (blue curve) andDUSP6mRNA level in KYSE-520 subcutaneous tumor samples(red curve) from mice treated with vehicle or IACS-13909. Plasma and tumor samples were harvested 24 hours after a single-dose treatment. N ¼ 3 mice/group/timepoint.D–F,Antitumor efficacy of IACS-13909 on MV-4-11 orthotopic mouse model. Mice were injected with MV-4-11-Luc cells through tail vein and treatedwith different doses of IACS-13909 once per day orally. N ¼ 10 mice/group. D, Representative mouse images from bioluminescence imaging indicating tumorvolume on day 34. E, Quantitated tumor volume determined by bioluminescence imaging. F, Kaplan–Meier curve showing the overall survival of the mice withor without IACS-13909 treatment. The dotted vertical line indicates when dosing stopped.

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CS cells demonstrated significantly reduced sensitivity toward osi-mertinib in an in vitro clonogenic assay, compared with the parentalcells (Fig. 3A). Importantly, IACS-13909 potently suppressedthe proliferation of both the parental cells and NCI-H1975 CScells in a dose-dependent manner, with similar potency (GI50�1 mmol/L; Fig. 3B). Consistent with the proliferation data, osimer-tinib at up to 300 nmol/L failed to suppress the levels of pERK orpEGFR inNCI-H1975CS cells (Fig. 3C), although in the parental cells,osimertinib at 10 nmol/L was sufficient for potent suppression ofpERK and pEGFR (12). Unlike osimertinib, IACS-13909 suppressedpERK in NCI-H1975 CS cells in a dose-dependent manner. As

expected, treatment with IACS-13909 did not reduce pEGFR becauseSHP2 is downstream of EGFR (Fig. 3C). Together, these data dem-onstrate that IACS-13909 suppresses the proliferation and MAPKpathway signaling in osimertinib-resistant cells harboring an EGFR-dependent resistance mutation in vitro.

To confirm the activity of IACS-13909 in human primary cancercells, we used the LD1-0025-200717 model, which is a PDX modelestablished from the hydrothorax of a patient with NSCLC whoprogressed on treatment with osimertinib. The tumor model harborsEGFRex19del/T790M/C797S, and is resistant to erlotinib, osimertinib, andthe combination of the two agents in vivo (28). To rapidly assess the

Figure 3.

IACS-13909 suppresses the proliferation and MAPK pathway signaling of EGFR TKI-resistant EGFRmut NSCLC models harboring an EGFR-dependent resistancemutation. A and B, Antiproliferative activity of osimertinib (Osi; A) and IACS-13909 (B) in NCI-H1975 parental and NCI-H1975 CS cells, determined by a 14-dayclonogenic assay. The NCI-H1975 parental cells harbor EGFRL858R/T790M, and the NCI-H1975 CS cells harbor EGFRL858R/T790M/C797S, where the C797S mutation onEGFR was introduced through CRISPR.N¼ 3. Confirmation of the C797Smutation is provided in Supplementary Fig. S2. C, The impact of osimertinib or IACS-13909on pERK1/2T202/Y204 and pEGFRY1068 levels in NCI-H1975 CS cells in vitro. Cells were treated with IACS-13909 or osimertinib for 2 hours and processed forWestern blotting. D and E, Antiproliferative activity of osimertinib (D) and IACS-13909 (E) on primary cells isolated from NSCLC PDX LD1-0025-200717 harboringEGFRex19del/T790M/C797S, determined by a 6-day ex vivo spheroid assay. The dotted horizontal line indicates the relative viable cell number when compound wasadded. N¼ 4. F, Tumor growth curve of the NCI-H1975 parental subcutaneous xenograft model in mice when treated with either vehicle, erlotinib at 10 mg/kg onceper day, osimertinib at 5mg/kg once per day, or IACS-13909 at 70 mg/kg once per day orally for 21 days. N ¼ 10 mice per group. Two-way ANOVA was used tocompare the growth curve of IACS-13909–treated tumors versus vehicle-treated tumors. �� , P < 0.01. G, Tumor growth curve of the NCI-H1975 CSsubcutaneous xenograft model in mice when treated with either vehicle, osimertinib at 5 mg/kg once per day, or IACS-13909 at 70 mg/kg once per dayorally for 12 days. The dotted vertical line denotes the final dose. N ¼ 10 mice per group. Two-way ANOVA was used to compare the growth curve ofIACS-13909–treated tumors versus osimertinib-treated tumors and vehicle-treated tumors. �� , P < 0.01.






antiproliferative effect of IACS-13909 in this model, we conducted anex vivo spheroid proliferation assay with cells freshly isolated fromtumors grown in mice. As expected, osimertinib treatment at 300nmol/L, a concentration that is approximately 40-fold higher than theGI50 of osimertinib in NCI-H1975 cells that harbor EGFRL858R/T790M

(Fig. 3A), had little impact on proliferation of the LD1-0025-200717spheroids ex vivo (Fig. 3D), confirming that this model is resistant toosimertinib. In contrast, IACS-13909 demonstrated dose-dependentsuppression of proliferation of the LD1-0025-200717 spheroidsex vivo, with GI50 approximately 1 mmol/L (Fig. 3E), similar to theGI50 of IACS-13909 in the NCI-H1975 cells. These data demonstratethe activity of IACS-13909 in primary cells derived from an osimerti-nib-resistant EGFRex19del/T790M/C797S PDX ex vivo.

To determine the in vivo activity of IACS-13909 in tumors har-boring an EGFR-dependent resistance mutation, we tested IACS-13909 in the NCI-H1975 parental and NCI-H1975 CS subcutaneousxenograft models in mice. As expected, in the NCI-H1975 parentaltumor harboring EGFRL858R/T790M, erlotinib treatment at 10 mg/kgonce per day delivered orally failed to suppress tumor proliferation,and treatment with osimertinib at 5 mg/kg once per day causedregression of the established tumor. Treatment with IACS-13909 at70 mg/kg once per day demonstrated robust antitumor efficacy, withtumor regression observed (Fig. 3F). In mice bearing the NCI-H1975CS tumors harboring EGFRL858R/T790M/C797S (Fig. 3G), treatment withosimertinib demonstrated little antitumor efficacy, which was distinctfrom the response observed in the parental tumors, confirming thatthis model is resistant to osimertinib in vivo. Importantly, treatmentwith IACS-13909 at 70 mg/kg once per day also demonstrated robustantitumor efficacy in theNCI-H1975CSmodel, with tumor regressionobserved. Together, these data demonstrate robust activity of IACS-13909 in osimertinib-resistant tumors harboring an EGFR-dependentresistance mutation in vivo.

In cells harboring EGFR-independent resistance mechanisms,IACS-13909 single agent or in combination with osimertinibsuppresses proliferation and MAPK pathway signaling in vitro

Beyond EGFR-specific mutations, a major resistance mechanismobserved with multiple generations of EGFRis is RTK-bypass,that is, the compensatory activation of alternate RTKs that main-tains downstream activation of the MAPK pathway with EGFRinhibited (14, 29–31). Prompted by the antiproliferative effect ofIACS-13909 in many RTK-activated human cancer cell lines, wetested the activity of IACS-13909 in EGFRmut NSCLC cells withRTK-bypass. We generated the HCC4006-osimertinib–resistant(OsiR) model by culturing HCC4006 cells harboring EGFRex19del

in the presence of 1 mmol/L osimertinib for an extended period oftime (�3 months), and confirmed the reduced osimertinib sensi-tivity in the OsiR derivative (Fig. 4A). We further demonstratedthat the HCC4006-OsiR cells did not harbor resistance mutations inEGFR (i.e., T790M, C797S, etc.) and had decreased pEGFR com-pared with the parental cells, suggesting that these cells wereswitching to other oncogenic drivers (Fig. 4B). Indeed, theHCC4006-OsiR cells demonstrated increased expression of multi-ple RTKs, including FGFR1, Axl, PDGFR, and IGF1Rb. The OsiRcells had also undergone EMT, with decreased expression ofepithelial marker, E-cadherin, and increased expression of mesen-chymal markers, vimentin and Zeb1 (Fig. 4B).

We conducted in vitro proliferation assays with HCC4006 parentaland HCC4006-OsiR cells, which were treated with IACS-13909either as a single agent or in combination with osimertinib.Despite the reduced osimertinib sensitivity observed in the OsiR cells,

IACS-13909 showed comparable single-agent antiproliferative effectin the parental and OsiR cells in clonogenic assays (Fig. 4C).Importantly, treatment with the combination of IACS-13909 andosimertinib resulted in a synergistic antiproliferative effect in bothmodels (Fig. 4D and E; Supplementary Fig. S3A and S3B), withpositive bliss scores in the majority of the concentrations tested(Supplementary Fig. S3C and S3D).

To understand the mechanism of action underlying the antiproli-ferative effect, signaling analysis was conducted in HCC4006-OsiRcells treated with osimertinib and/or IACS-13909 in vitro. The MPAS(MAPK pathway activity score) signature is composed of 10 genes thatreflects MAPK pathway activity (27). On the basis of the MPASsignature, a 13-gene signature (“MPAS-plus”) was developed, whichincludes three additional MAPK-targeted genes (ETV1, EGR1, andFOSL1; refs. 32, 33). Osimertinib alone failed to potently suppressMAPK pathway signaling in the HCC4006-OsiR cells, as demonstrat-ed by lack of suppression of DUSP6 mRNA levels and other MPAS-plus genes. In contrast, IACS-13909 potently suppressed MAPKpathway signaling, both as a single agent and in combination withosimertinib (Fig. 4F). The suppression was achieved with 2-hour, 48-hour, and 7-day treatment of IACS-13909, suggesting sustainedsuppression of MAPK pathway, despite the observed partial adapta-tion (less suppression with prolonged treatment compared with acutetreatment). This is consistent with the notion that SHP2 inhibitionsuppresses the signaling downstream of multiple RTKs, therefore,delaying the multi-RTK–mediated rapid adaptation that is com-monly observed toward MAPK pathway inhibitors (34–36). It isalso noteworthy that treatment with the combination of IACS-13909 and osimertinib did not cause further suppression of theMAPK pathway compared with IACS-13909 single agent in vitro,suggesting potential additional non-MAPK–mediated mechanismsfor the synergistic antiproliferative effect between osimertinib andIACS-13909 in the in vitro setting.

Combination of IACS-13909 and osimertinib extends thedurability of osimertinib response in osimertinib-sensitivetumors and causes tumor regression in osimertinib-resistanttumors with RTK-bypass

Our in vitro data with IACS-13909, either as single agent or incombination with osimertinib, in the HCC4006-OsiR model thatharbors EGFR-independent resistance mechanisms, prompted us toconduct further evaluation in vivo. The osimertinib-resistant EGFRmut

NSCLCHCC827-ER1 cells were generated by exposing HCC827 cells,which harbor an EGFR-activating mutation (EGFRex19del), to erlotinibin culture (20). The HCC827-ER1 cells do not harbor EGFR-dependent resistance mutations, but do have amplified c-MET, agenetic alteration observed in tumors from patients who have relapsedon erlotinib and osimertinib (14, 29). The HCC827-ER1 cells areresistant to erlotinib and also to osimertinib (20).

In the osimertinib-sensitive HCC827 xenograft model (Fig. 5A),IACS-13909 at 70 mg/kg dosed daily as a single agent, potentlysuppressed tumor growth, leading to tumor stasis, and osimertinibdosed as a single agent at 5 mg/kg once per day caused robust tumorregression. As expected, treatment with the combination of IACS-13909 and osimertinib yielded tumor regression, similar to thatobserved with osimertinib alone, during the period of compoundadministration. However, following cessation of dosing, tumors inmice treated with the combination did not grow, whereas those treatedwith osimertinib showed significant growth beginning approximately30 days after the final dose. Importantly, the combination treatment inHCC827 xenograft model was tolerated, as shown by the maintenance

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of body weight during the study (<10% average body weight loss;Supplementary Fig. S4A). Thus, treatment with the combination ofIACS-13909 and osimertinib resulted in a more durable antitumorresponse (Fig. 5A), consistent with the in vitro observation in theHCC4006 cells (Fig. 4D).

In the osimertinib-resistant HCC827-ER1 model (Fig. 5B), tumorsin mice treated with IACS-13909 at 70 mg/kg once per day demon-strated tumor stasis, similar to the response in the HCC827 parentalmodel. However, tumors inmice treatedwith osimertinib continued to

grow on treatment, indicating reduced osimertinib sensitivity inHCC827-ER1 as compared with the HCC827 model. Importantly,the combination treatment of IACS-13909 and osimertinib causedrobust regression of the HCC827-ER1 tumor, similar to the single-agent effect of osimertinib in the parental model. Waterfall plot oftumor volume demonstrated that almost all mice treated with thecombination for 3 weeks had tumor regression (Fig. 5C). The com-bination of osimertinib and IACS-13909 in the HCC827-ER1 xeno-graft model was also tolerated, as shown by the maintenance of body

Figure 4.

IACS-13909 suppresses the prolif-eration and MAPK pathwaysignaling of osimertinib-resistantEGFRmut NSCLC cells harboringRTK-bypass in vitro. A, Activity ofosimertinib in EGFRmut HCC4006and HCC4006-OsiR models,determined by clonogenic assays.N¼ 3. B, The level of various RTKsand EMT markers in HCC4006parental (Par) and OsiR models.The OsiR models were maintainedin the presence of 1 mmol/L osi-mertinib. To generate samplesfor signaling analysis, cells werecultured in the absence of osi-mertinib for 1 day, then treatedwith DMSO or 1 mmol/L osimerti-nib for 2 hours before being har-vested and processed for West-ern blotting. C, Activity of IACS-13909 in EGFRmut HCC4006and HCC4006-OsiR models,determined by in vitro clonogenicassays. N ¼ 4–8. D and E, Anti-proliferative activity of IACS-13909 in combination with osi-mertinib in HCC4006 (D) andHCC4006-OsiR (E) cell lines,determined by in vitro clonogenicassays. Percent of inhibition, cal-culated from quantitated cellnumber, is provided inSupplemen-tary Fig. S3A and S3B. Bliss scorecalculation is provided in Supple-mentary Fig. S3C and S3D. N ¼ 4.F, Heatmap from gene expressionanalysis showing the impact ofosimertinib (100 nmol/L) andIACS-13909 (3 mmol/L) either assingle agent or in combination ona MAPK signature (MPAS-plussignature) in HCC4006-OsiR cellsover a time course in vitro. N ¼ 3.FC, fold change.






Figure 5.

Antitumor efficacy of treatment with IACS-13909 and osimertinib (osi), alone and in combination (combo), in an MET-amplified EGFRi acquired–resistant modelin vivo.A andB,Tumorgrowth curves of EGFRmutHCC827 (A) andHCC827-ER1 (B) xenograftmodels treatedwith vehicle (0.5%methylcellulose, onceper dayþ0.5%HPMC, once per day), osimertinib (0.5%methylcellulose, once per dayþ osimertinib 5mg/kg, once per day), IACS-13909 (IACS-13909 70 or 80mg/kg, once per dayþ0.5% HPMC, once per day), or the combination (IACS-13909 70 or 80mg/kg, once per dayþ osimertinib 5mg/kg, once per day).N¼ 10mice per group (A).N ≥ 10mice for all groups (B). In bothA andB, the graphs represent pooled data from two independent experiments. In one experiment, IACS-13909was used at 70mg/kgand in the other IACS-13909 was used at 80 mg/kg. Two-way ANOVA was used to compare the tumor growth curves of osimertinib single-agent group versus thecombination group. ��, P < 0.01. C, Relative tumor volume change on day 22 from data shown in B, when dosing ended. The tumor volume of each mouse wasnormalized to the tumor volume when dosing started. 0 indicates tumor stasis; <0 indicates tumor regression. D, Antitumor efficacy of treatment with thecombination of IACS-13909 þ osimertinib on HCC827-ER1 tumors that outgrew on osimertinib treatment. When average tumor volume reached 300 mm3, micebearing HCC827-ER1 tumorswere treatedwith vehicle (n¼ 5) or osimertinib (5mg/kg, once per day, n¼ 10).When tumors progressed on osimertinib treatment andreached 500 mm3, the treated tumors were rerandomized and subjected to treatment with osimertinib (5 mg/kg, once per day; n ¼ 5) or the combination(osimertinib 5 mg/kg, once per dayþ IACS-13909 70 mg/kg, once per day; n ¼ 5). Two-way ANOVA was used to compare osimertinib single-agent group versuscombination group. �� ,P<0.01. E,Modulation ofDUSP6mRNA levels in HCC827-ER1 subcutaneous tumors during a 24-hour time period following 1 day treatment, asconducted in B. Tumor samples were harvested at 6, 8, and 24 hours. DUSP6mRNA levels were determined by qRT-PCR. N¼ 4 mice per group for each timepoint.Two-tail t test was conducted to compare the combination group versus vehicle or single-agent groups. � , P < 0.05; �� , P < 0.01. F, Gene expression analysis wasconductedwith tumor samples harvested at 24 hours after SHP2 inhibitor/18 hours after osimertinib timepoint inE. Modulation of aMAPK-pathway signature (MPAS-plus) by osimertinib, IACS-13909, and the combination is shown. FC, fold change.

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weight during the study (≤5% body weight loss; SupplementaryFig. S4B). This result demonstrates that addition of the SHP2 inhibitor,IACS-13909, leads to resensitization of the osimertinib-resistantHCC827-ER1 model harboring RTK-bypass to treatment withosimertinib.

We further determined whether combination of osimertinib andIACS-13909 can inhibit the growth of HCC827-ER1 tumors thatprogressed on osimertinib treatment. We started osimertinib single-agent treatment when average tumor volume was approximately 300mm3; the tumors progressed and average tumor volume reachedapproximately 500 mm3 (67% increase in tumor volume) within3 weeks. At this time, the osimertinib-treatedmice were rerandomizedand enrolled into treatment with osimertinib alone or with thecombination of osimertinib and IACS-13909. Whereas tumors inmice treated with osimertinib alone continued to grow, tumors inmice treated with the combination of osimertinib and IACS-13909demonstrated significantly inhibited tumor growth, with tumorregression (Fig. 5D).

To gain insight into the mechanism of the combination treatment,we analyzed HCC827-ER1 tumors frommice treated with osimertinibalone, IACS-13909 alone, or the combination. Mice were dosedfollowing the same schedule as in the efficacy study, with a 6-hourinterval between IACS-13909 dosing and osimertinib dosing (Fig. 5E),and samples were harvested at three timepoints during the 24-hourdosing cycle. In the HCC827-ER1 tumors, osimertinib treatmentmodestly suppressed DUSP6 mRNA levels. IACS-13909 potentlysuppressed DUSP6 mRNA levels at 6- and 8-hour after treatment,with the extent of suppression decreased afterwards. Importantly, thecombined treatment with the two compounds maintained the potentsuppression of DUSP6 mRNA levels throughout the 24-hour dosingcycle (Fig. 5E). To further confirm the combinational effect onMAPKpathway signaling, we evaluated the MPAS-plus panel (Fig. 5F). Atclose to dosing trough, while each monotherapy had little effect on themRNA levels of the genes on the panel, the combination of IACS-13909 and osimertinib more potently suppressed the level of most ofthe MPAS-plus genes. These data suggest that combined treatmentwith osimertinib and IACS-13909 potently suppressesMAPKpathwaysignaling, to a larger extent than either single agent, in an osimertinib-resistant model with RTK-bypass, consistent with the efficacy data.

DiscussionIn this study, we report the discovery of IACS-13909, a potent and

selective allosteric SHP2 inhibitor. Our in vitro and in vivo datademonstrate that IACS-13909 has antitumor activity and suppressesMAPK pathway signaling in RTK-dependent cancers. Importantly,IACS-13909 exhibits antitumor efficacy in osimertinib-resistant mod-els that harbor clinically relevant resistance mechanisms. In osimerti-nib-resistant tumorswith EGFR-dependent resistancemutations, suchas the C797S mutation in the NCI-H1975 CS cells, EGFR remains theprimary oncogenic driver and signals through SHP2. Thus, althoughosimertinib is not able to potently suppress EGFR here, inhibition ofSHP2, which lies downstream of EGFR, blocks signaling through theMAPK pathway (Fig. 6A). In osimertinib-resistant tumors, in whichthe MAPK pathway is activated because of activation of an alternateRTK, such asMET in theHCC827-ER1 cells, the alternate RTK signalsthrough SHP2 to maintain the MAPK pathway activity (Fig. 6B).Together, our preclinical data demonstrate that the SHP2 inhibitor,IACS-13909, is effective in overcoming both EGFR-dependent andEGFR-independent resistance mechanisms toward osimertinib.Importantly, the ability of SHP2 inhibition in targeting multipleresistance mechanisms is anticipated to address the heterogeneity andplasticity of osimertinib resistance.

Amajor challenge in targeting the RTK/MAPK pathway is acquiredresistance, whereby a tumor initially responds to treatment, butregrows on continued treatment. This can be attributed to both theadaptability of the cancer cells and the heterogeneity of the primarytumors. Cancer cells very often harbor a primary oncogenic driver.When the primary driver is blocked, other oncogenic drivers eitherwithin the same cells or from a different clone emerge as the alternatedriving force for tumor growth. Combining one drug targeting theprimary oncogenic driver and a second drug suppressing multiplepotential alternative drivers is an attractive strategy. To improvethe therapeutic index, ideally the first drug should be mutant selective,and to ensure broad targeting of potential secondary drivers, thesecond drug should target WT protein as well. Here, we proposecombining EGFR-mutant selective inhibitor, osimertinib (12), andSHP2 allosteric inhibitor, IACS-13909, that is not mutant selective. Attolerated doses, such combination achieves more durable responsecompared with osimertinib single agent in osimertinib-sensitive

Figure 6.

Proposed model for IACS-13909 in overcoming both EGFR-dependent and EGFR-independent osimertinib resistance mechanisms. A, In tumors harboring an EGFRmutation that confers resistance toosimertinib (e.g., C797S), EGFR remains as theprimaryoncogenic driver and signals throughSHP2. SHP2 inhibitionby an allostericSHP2 inhibitor such as IACS-13909 is effective in inhibiting proliferation of the tumor.B, In tumorswhere inhibition of EGFR results in compensatory activation of oneor more RTKs (“RTK-bypass”), a SHP2 inhibitor can inhibit tumor cell proliferation by blocking signaling downstream of the activated RTKs.






EGFRmut NSCLC tumors, and causes tumor regression in osimertinib-resistant EGFRmut NSCLC xenograft tumors in mice.

Our data demonstrate that IACS-13909 has antitumor activity incancers with a broad range of RTKs as the oncogenic driver. While weprovide data showing SHP2 inhibition can overcome both EGFR-dependent and EGFR-independent osimertinib resistance in EGFRmut

NSCLC, a SHP2 inhibitor can be usedmore broadly. Several additionalcombination strategies with an allosteric SHP2 inhibitor have beenproposed in overcoming resistance to targeted agents. First, in ALKinhibitor–resistant preclinical models with RTK-bypass as a resistancemechanism, in vivo efficacy for the treatment with combination ofALK inhibitor, ceritinib, and SHP099 has been reported (37). Second,the clinical response toMEK inhibitors is limited by adaptive feedbackactivation through multiple RTKs (35, 38), therefore, combination ofSHP2 inhibitor and MEK inhibitor has demonstrated antitumorefficacy in mice (8, 36). In addition to MEK inhibitor, preclinical datafor combining ERK inhibitor have been reported (39). A majorchallenge with combining SHP2 inhibitor and MEK inhibitor (orERK inhibitor) when both molecules target WT enzymes is thetherapeutic index. SHP2 inhibitors were used at reduced doses ordosing frequencies in both combination strategies inmice (36, 39). It isspeculated that sustained shutdown of the MAPK pathway in normaltissue may not be tolerated, therefore, reduced dose or dosing fre-quency that leads to pulsatile shutdown of the MAPK pathway had tobe performed. Most recently, multiple approaches have identifiedcombining a KRASG12C-mutant–specific inhibitor and SHP2 inhibitoras a strategy for achieving more robust and durable response (40, 41).Identifying the most optimal combination strategy for a SHP2 allo-steric inhibitor requires additional preclinical work and, most impor-tantly, clinical trials. Currently, several SHP2 allosteric inhibitors(TNO155, RMC-4630, JAB-3068, JAB-3312, andRLY-1971) are underearly-phase clinical development. An advanced derivative of IACS-13909 will enter phase I clinical trial in later 2020.

Disclosure of Potential Conflicts of InterestY. Sun reports other funding fromNavire Pharma (this workwas funded byNavire

Pharma Inc., a BridgeBio Company. The University of Texas MD Anderson CancerCenter and Navire Pharma, Inc. are parties to a collaboration and license pursuant towhich MD Anderson and Navire will collaborate on the conduct of research anddevelopment of products. Under this agreement, the Board of Regents of TheUniversity of Texas System, on behalf of MD Anderson, received equity, milestonepayments, and royalties in Navire. Proceeds may be distributed based on UT SystemIntellectual Property policy) during the conduct of the study. B.A. Meyers reportsother funding fromNavire Pharma Inc. (this work was funded byNavire Pharma Inc.,a BridgeBio Company. The University of Texas MD Anderson Cancer Center andNavire Pharma, Inc. are parties to a collaboration and license pursuant to which MDAnderson and Navire will collaborate on the conduct of research and development ofproducts. Under this agreement, the Board of Regents of The University of TexasSystem, on behalf of MD Anderson, received equity, milestone payments, androyalties in Navire. Proceeds may be distributed based on UT System IntellectualProperty policy) during the conduct of the study. B. Czako reports other funding fromNavire Pharma (this work was funded by Navire Pharma Inc., a BridgeBio Company.The University of Texas MD Anderson Cancer Center and Navire Pharma, Inc. areparties to a collaboration and license pursuant towhichMDAnderson andNavire willcollaborate on the conduct of research and development of products. Under thisagreement, the Board of Regents of The University of Texas System, on behalf of MDAnderson, received equity, milestone payments, and royalties in Navire. Proceedsmay be distributed based on UT System Intellectual Property policy) during theconduct of the study, as well as has a patent for WO 2020033828 A1 20200213 issuedand a patent for US 20170342078 A1 20171130 issued. P. Leonard reports otherfunding from Navire Pharma Inc. (this work was funded by Navire Pharma Inc., aBridgeBio Company. The University of Texas MD Anderson Cancer Center andNavire Pharma, Inc. are parties to a collaboration and license pursuant to which MDAnderson and Navire will collaborate on the conduct of research and development ofproducts. Under this agreement, the Board of Regents of The University of Texas

System, on behalf of MD Anderson, received equity, milestone payments, androyalties in Navire. Proceeds may be distributed based on UT System IntellectualProperty policy) during the conduct of the study, as well as has a patent for US10280171 issued and licensed to Navire Pharma Inc. F. Mseeh reports other fundingfrom Navire Pharma Inc. (this work was funded by Navire Pharma Inc., a BridgeBioCompany. TheUniversity of TexasMDAndersonCancer Center andNavire Pharma,Inc. are parties to a collaboration and license pursuant to which MD Anderson andNavire will collaborate on the conduct of research and development of products.Under this agreement, the Board of Regents of The University of Texas System, onbehalf ofMDAnderson, received equity, milestone payments, and royalties inNavire.Proceeds may be distributed based on UT System Intellectual Property policy) duringthe conduct of the study, as well as has a patent for US 10280171 issued and licensed toNavire Pharma Inc. C.A. Parker reports other funding from Navire Pharma Inc. (thiswork was funded by Navire Pharma Inc., a BridgeBio Company. The University ofTexas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to acollaboration and license pursuant to which MD Anderson and Navire will collab-orate on the conduct of research and development of products. Under this agreement,the Board of Regents of The University of Texas System, on behalf of MD Anderson,received equity, milestone payments, and royalties in Navire. Proceeds may bedistributed based on UT System Intellectual Property policy) during the conductof the study, as well as has a patent for US10280171B2 issued. J.B. Cross reports otherfunding from Navire Pharma Inc. (this work was funded by Navire Pharma Inc., aBridgeBio Company. The University of Texas MD Anderson Cancer Center andNavire Pharma, Inc. are parties to a collaboration and license pursuant to which MDAnderson and Navire will collaborate on the conduct of research and development ofproducts. Under this agreement, the Board of Regents of The University of TexasSystem, on behalf of MD Anderson, received equity, milestone payments, androyalties in Navire. Proceeds may be distributed based on UT System IntellectualProperty policy) during the conduct of the study, as well as has a patent forWO2020033828 pending and licensed to Navire Pharma and a patent for US10280171 issued and licensed to Navire Pharma. M.E. Di Francesco reports otherfunding from Navire Pharma Inc. (this work was funded by Navire Pharma Inc., aBridgeBio Company. The University of Texas MD Anderson Cancer Center andNavire Pharma, Inc. are parties to a collaboration and license pursuant to which MDAnderson and Navire will collaborate on the conduct of research and development ofproducts. Under this agreement, the Board of Regents of The University of TexasSystem, on behalf of MD Anderson, received equity, milestone payments, androyalties in Navire. Proceeds may be distributed based on UT System IntellectualProperty policy) during the conduct of the study and personal fees from Immuno-Genesis, Inc. (consultant for organic synthesis and medicinal chemistry) outside thesubmitted work. B.J. Bivona reports other funding from Navire Pharma Inc. (thiswork was funded by Navire Pharma Inc., a BridgeBio Company. The University ofTexas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to acollaboration and license pursuant to which MD Anderson and Navire will collab-orate on the conduct of research and development of products. Under this agreement,the Board of Regents of The University of Texas System, on behalf of MD Anderson,received equity, milestone payments, and royalties in Navire. Proceeds may bedistributed based on UT System Intellectual Property policy) during the conductof the study. C.A. Bristow reports other funding from Navire Pharma Inc. (this workwas funded by Navire Pharma Inc., a BridgeBio company. The University of TexasMD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaborationand license pursuant to which MD Anderson and Navire will collaborate on theconduct of research and development of products. Under this agreement, the Board ofRegents of The University of Texas System, on behalf of MD Anderson, receivedequity, milestone payments, and royalties in Navire. Proceeds may be distributedbased onUT System Intellectual Property policy) during the conduct of the study. J.P.Burke reports other funding from MD Anderson (this work was funded by NavirePharma Inc., a BridgeBio Company. The University of Texas MD Anderson CancerCenter and Navire Pharma, Inc. are parties to a collaboration and license pursuant towhich MD Anderson and Navire will collaborate on the conduct of research anddevelopment of products. Under this agreement, the Board of Regents of TheUniversity of Texas System, on behalf of MD Anderson, received equity, milestonepayments, and royalties in Navire. Proceeds may be distributed based on UT SystemIntellectual Property policy) during the conduct of the study and MD Anderson(salary paid during prior time until July 2018 when no longer employee) outside thesubmitted work, as well as has a patent for WO2019213318 A1 issued (inventor onrelevant patent). C.L. Carroll reports other funding from Navire Pharma Inc., aBridgeBio Company (this work was funded by Navire Pharma Inc., a BridgeBioCompany. TheUniversity of TexasMDAndersonCancer Center andNavire Pharma,Inc. are parties to a collaboration and license pursuant to which MD Anderson andNavire will collaborate on the conduct of research and development of products.

Sun et al.





Under this agreement, the Board of Regents of The University of Texas System, onbehalf ofMDAnderson, received equity, milestone payments, and royalties inNavire.Proceeds may be distributed based on UT System Intellectual Property policy) duringthe conduct of the study, as well as has a patent forWO/2020/033828 issued to Boardof Regents, TheUniversity of Texas System.G.Gao reports other funding fromNavirePharma Inc. (thisworkwas funded byNavire Pharma Inc., a BridgeBioCompany. TheUniversity of TexasMDAnderson Cancer Center andNavire Pharma, Inc. are partiesto a collaboration and license pursuant to which MD Anderson and Navire willcollaborate on the conduct of research and development of products. Under thisagreement, the Board of Regents of The University of Texas System, on behalf of MDAnderson, received equity, milestone payments, and royalties in Navire. Proceedsmay be distributed based on UT System Intellectual Property policy) during theconduct of the study. S. Gera reports other funding from Navire Pharma Inc. (thiswork was funded by Navire Pharma Inc., a BridgeBio Company. The University ofTexas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to acollaboration and license pursuant to which MD Anderson and Navire will collab-orate on the conduct of research and development of products. Under this agreement,the Board of Regents of The University of Texas System, on behalf of MD Anderson,received equity, milestone payments, and royalties in Navire. Proceeds may bedistributed based on UT System Intellectual Property policy) during the conductof the study. V. Giuliani reports other funding from Navire Pharma Inc., a BridgeBioCompany (the University of TexasMDAnderson Cancer Center andNavire Pharma,Inc. are parties to a collaboration and license pursuant to which MD Anderson andNavire will collaborate on the conduct of research and development of products.Under this agreement, the Board of Regents of The University of Texas System, onbehalf ofMDAnderson, received equity, milestone payments, and royalties inNavire.Proceeds may be distributed based on UT System Intellectual Property policy) duringthe conduct of the study. J.K. Huang reports grants, nonfinancial support, and otherfunding from Navire Pharma Inc. (this work was funded by Navire Pharma Inc., aBridgeBio Company. The University of Texas MD Anderson Cancer Center andNavire Pharma, Inc. are parties to a collaboration and license pursuant to which MDAnderson and Navire will collaborate on the conduct of research and development ofproducts. Under this agreement, the Board of Regents of The University of TexasSystem, on behalf of MD Anderson, received equity, milestone payments, androyalties in Navire) during the conduct of the study. J.J. Kovacs reports other fundingfrom Navire Pharma (this work was funded by Navire Pharma Inc., a BridgeBioCompany. TheUniversity of TexasMDAndersonCancer Center andNavire Pharma,Inc. are parties to a collaboration and license pursuant to which MD Anderson andNavire will collaborate on the conduct of research and development of products.Under this agreement, the Board of Regents of The University of Texas System, onbehalf ofMDAnderson, received equity, milestone payments, and royalties inNavire.Proceeds may be distributed based on UT System Intellectual Property policy) duringthe conduct of the study. C.-Y. Liu reports other funding from Navire Pharma (thiswork was funded by Navire Pharma Inc., a BridgeBio Company. The University ofTexas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to acollaboration and license pursuant to which MD Anderson and Navire will collab-orate on the conduct of research and development of products. Under this agreement,the Board of Regents of The University of Texas System, on behalf of MD Anderson,received equity, milestone payments, and royalties in Navire. Proceeds may bedistributed based on UT System Intellectual Property policy) during the conductof the study. A.M. Lopez reports other funding from Navire Pharma Inc. (this workwas funded by Navire Pharma Inc., a BridgeBio Company. The University of TexasMD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaborationand license pursuant to which MD Anderson and Navire will collaborate on theconduct of research and development of products. Under this agreement, the Board ofRegents of The University of Texas System, on behalf of MD Anderson, receivedequity, milestone payments, and royalties in Navire. Proceeds may be distributedbased on UT System Intellectual Property policy) during the conduct of the study.X. Ma reports other funding from Navire Pharma Inc., a BridgeBio Company (TheUniversity of TexasMDAnderson Cancer Center andNavire Pharma, Inc. are partiesto a collaboration and license pursuant to which MD Anderson and Navire willcollaborate on the conduct of research and development of products. Under thisagreement, the Board of Regents of The University of Texas System, on behalf of MDAnderson, received equity, milestone payments, and royalties in Navire. Proceedsmay be distributed based on UT System Intellectual Property policy) during theconduct of the study. T. McAfoos reports other funding from Navire Pharma Inc.(this work was funded by Navire Pharma Inc., a BridgeBio Company. The Universityof Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to acollaboration and license pursuant to which MD Anderson and Navire will collab-orate on the conduct of research and development of products. Under this agreement,the Board of Regents of The University of Texas System, on behalf of MD Anderson,

received equity, milestone payments, and royalties in Navire. Proceeds may bedistributed based on UT System Intellectual Property policy) during the conductof the study, as well as has a patent for WO 2019213318 issued. M.A. Miller reportsother funding from Navire Pharma (this work was funded by Navire Pharma Inc., aBridgeBio Company. The University of Texas MD Anderson Cancer Center andNavire Pharma, Inc. are parties to a collaboration and license pursuant to which MDAnderson and Navire will collaborate on the conduct of research and development ofproducts. Under this agreement, the Board of Regents of The University of TexasSystem, on behalf of MD Anderson, received equity, milestone payments, androyalties in Navire. Proceeds may be distributed based on UT System IntellectualProperty policy) during the conduct of the study. M. Peoples reports other fundingfrom Navire Pharma (this work was funded by Navire Pharma Inc., a BridgeBioCompany. TheUniversity of TexasMDAndersonCancer Center andNavire Pharma,Inc. are parties to a collaboration and license pursuant to which MD Anderson andNavire will collaborate on the conduct of research and development of products.Under this agreement, the Board of Regents of The University of Texas System, onbehalf ofMDAnderson, received equity, milestone payments, and royalties inNavire.Proceeds may be distributed based on UT System Intellectual Property policy) duringthe conduct of the study. V. Ramamoorthy reports other funding fromNavire Pharma(this work was funded by Navire Pharma Inc., a BridgeBio Company. The Universityof Texas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to acollaboration and license pursuant to which MD Anderson and Navire will collab-orate on the conduct of research and development of products. Under this agreement,the Board of Regents of The University of Texas System, on behalf of MD Anderson,received equity, milestone payments, and royalties in Navire. Proceeds may bedistributed based on UT System Intellectual Property policy) during the conductof the study. N.D. Spencer reports other funding fromNavire Pharma Inc. (this workwas funded by Navire Pharma Inc., a BridgeBio Company. The University of TexasMD Anderson Cancer Center and Navire Pharma, Inc. are parties to a collaborationand license pursuant to which MD Anderson and Navire will collaborate on theconduct of research and development of products. Under this agreement, the Board ofRegents of The University of Texas System, on behalf of MD Anderson, receivedequity, milestone payments, and royalties in Navire. Proceeds may be distributedbased on UT System Intellectual Property policy) during the conduct of the study.E. Suzuki reports other funding fromNavire Pharma (this work was funded byNavirePharma Inc., a BridgeBio Company. The University of Texas MD Anderson CancerCenter and Navire Pharma, Inc. are parties to a collaboration and license pursuant towhich MD Anderson and Navire will collaborate on the conduct of research anddevelopment of products. Under this agreement, the Board of Regents of TheUniversity of Texas System, on behalf of MD Anderson, received equity, milestonepayments, and royalties in Navire. Proceeds may be distributed based on UT SystemIntellectual Property policy) during the conduct of the study. C.C. Williams reportsother funding from Navire Pharma (this work was funded by Navire Pharma Inc., aBridgeBio Company. The University of Texas MD Anderson Cancer Center andNavire Pharma, Inc. are parties to a collaboration and license pursuant to which MDAnderson and Navire will collaborate on the conduct of research and development ofproducts. Under this agreement, the Board of Regents of The University of TexasSystem, on behalf of MD Anderson, received equity, milestone payments, androyalties in Navire. Proceeds may be distributed based on UT System IntellectualProperty policy) during the conduct of the study. A.M. Zuniga reports other fundingfrom Navire Pharma (this work was funded by Navire Pharma Inc., a BridgeBioCompany. TheUniversity of TexasMDAndersonCancer Center andNavire Pharma,Inc. are parties to a collaboration and license pursuant to which MD Anderson andNavire will collaborate on the conduct of research and development of products.Under this agreement, the Board of Regents of The University of Texas System, onbehalf ofMDAnderson, received equity, milestone payments, and royalties inNavire.Proceeds may be distributed based on UT System Intellectual Property policy) duringthe conduct of the study. G.F. Draetta reports other funding fromNavire Pharma (thiswork was funded by Navire Pharma Inc., a BridgeBio Company. The University ofTexas MD Anderson Cancer Center and Navire Pharma, Inc. are parties to acollaboration and license pursuant to which MD Anderson and Navire will collab-orate on the conduct of research and development of products. Under this agreement,the Board of Regents of The University of Texas System, on behalf of MD Anderson,received equity, milestone payments, and royalties in Navire. Proceeds may bedistributed based on UT System Intellectual Property policy) during the conductof the study and personal fees from Taiho Pharmaceutical Co., Blueprint Medicines,BiovelocITA (stock ownership), Metabomed (stock ownership), Nurix, Inc.(stock ownership), Orionis Biosciences (stock ownership), Frontier Medicines (stockownership), and Forma Therapeutics (stock ownership) outside the submitted work.J.R. Marszalek reports other funding from Navire Pharma (sponsored research)during the conduct of the study. T.P. Heffernan reports other funding from Navire






Pharma Inc. (thisworkwas funded byNavire Pharma Inc., a BridgeBioCompany. TheUniversity of TexasMDAnderson Cancer Center andNavire Pharma, Inc. are partiesto a collaboration and license pursuant to which MD Anderson and Navire willcollaborate on the conduct of research and development of products. Under thisagreement, the Board of Regents of The University of Texas System, on behalf of MDAnderson, received equity, milestone payments, and royalties in Navire. Proceedsmay be distributed based on UT System Intellectual Property policy) during theconduct of the study and personal fees and other compensation from Cullgen Inc.(stock ownership and advisory fees) outside the submitted work. P. Jones reportsother funding fromNavire Pharma Inc (this work was funded by Navire Pharma Inc.,a BridgeBio Company. The University of Texas MD Anderson Cancer Center andNavire Pharma, Inc. are parties to a collaboration and license pursuant to which MDAnderson and Navire will collaborate on the conduct of research and development ofproducts. Under this agreement, the Board of Regents of The University of TexasSystem, on behalf of MD Anderson, received equity, milestone payments, androyalties in Navire. Proceeds may be distributed based on UT System IntellectualProperty policy) during the conduct of the study, as well as has a patent forWO2020033828 pending and licensed to Navire Pharma Inc. and a patent for US10280171 issued and licensed to Navire Pharma Inc. No potential conflicts of interestwere disclosed by the other authors.

Authors’ ContributionsY. Sun: Conceptualization, data curation, formal analysis, supervision, validation,

investigation, visualization, methodology, writing-original draft, projectadministration, writing-review and editing. B.A. Meyers: Data curation, formalanalysis, validation, investigation, methodology. B. Czako: Conceptualization,supervision, investigation, project administration, writing-review and editing.P. Leonard: Formal analysis, supervision, investigation, visualization, methodology,writing-original draft,writing-reviewandediting.F.Mseeh:Supervision,methodology,writing-original draft. A.L. Harris: Validation, investigation, methodology. Q. Wu:Validation, investigation, methodology. S. Johnson: Validation, investigation,methodology. C.A. Parker: Validation, investigation, methodology. J.B. Cross:Supervision, writing-review and editing. M.E. Di Francesco: Resources, supervision.

B.J. Bivona: Methodology. C.A. Bristow: Formal analysis, supervision. J.P. Burke:Investigation. C.C. Carrillo: Investigation. C.L. Carroll: Investigation. Q. Chang:Investigation, methodology. N. Feng: Supervision. G. Gao: Investigation. S. Gera:Investigation. V. Giuliani: Supervision. J.K. Huang: Formal analysis, methodology,writing-original draft. Y. Jiang: Supervision, writing-original draft. Z. Kang:Investigation. J.J. Kovacs: Supervision, writing-review and editing. C.-Y. Liu:Investigation. A.M. Lopez: Methodology. X. Ma: Investigation. P.K. Mandal:Investigation. T. McAfoos: Investigation.M.A. Miller: Investigation. R.A. Mullinax:Investigation.M.Peoples: Supervision, investigation.V.Ramamoorthy: Investigation.S. Seth: Data curation, investigation. N.D. Spencer: Investigation. E. Suzuki:Investigation. C.C. Williams: Investigation. S.S. Yu: Investigation. A.M. Zuniga:Methodology. G.F. Draetta: Resources, supervision, funding acquisition.J.R. Marszalek: Resources, supervision. T.P. Heffernan: Resources, supervision,writing-review and editing. N.E. Kohl: Conceptualization, resources, supervision,project administration, writing-review and editing. P. Jones: Conceptualization,resources, supervision, funding acquisition, project administration, writing-reviewand editing.

AcknowledgmentsThe authors thank DV-MS at MDAnderson Cancer Center (MDACC) for mouse

husbandry and care, Research Histology, Pathology & Imaging Core at MDACCScience Park for histology service, and all members at TRACTION and the Institutefor Applied Cancer Science at MDACC for discussions. The authors also thankChemPartners, Eurofins, Crown Bioscience, LIDE Biotech, and the experimentaltherapeutics core at the Dana-Farber Cancer Institute for service.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 18, 2020; revised August 4, 2020; accepted September 1, 2020;published first September 14, 2020.

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2020;80:4840-4853. Published OnlineFirst September 14, 2020.Cancer Res Yuting Sun, Brooke A. Meyers, Barbara Czako, et al. toward OsimertinibEGFR-Dependent and EGFR-Independent Resistance Mechanisms Allosteric SHP2 Inhibitor, IACS-13909, Overcomes

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