allosteric inhibition of shp2 stimulates antitumor ... · allosteric inhibition of the protein...

CANCER RESEARCH | TUMOR BIOLOGYAND IMMUNOLOGY

Allosteric Inhibition of SHP2 Stimulates AntitumorImmunity by Transforming the ImmunosuppressiveEnvironment A C

Elsa Quintana1, Christopher J. Schulze1, Darienne R. Myers1, Tiffany J. Choy1, Kasia Mordec1, DavidWildes1,Nataliya Tobvis Shifrin1, Amira Belwafa1, Elena S. Koltun2, Adrian L. Gill2, Mallika Singh1, Stephen Kelsey1,2,Mark A. Goldsmith1,2, Robert Nichols1, and Jacqueline A.M. Smith1

ABSTRACT◥

The protein tyrosine phosphatase SHP2 binds to phosphorylatedsignaling motifs on regulatory immunoreceptors including PD-1,but its functional role in tumor immunity is unclear. Using pre-clinical models, we show that RMC-4550, an allosteric inhibitor ofSHP2, induces antitumor immunity, with effects equivalent to orgreater than those resulting from checkpoint blockade. In the tumormicroenvironment, inhibition of SHP2 modulated T-cell infiltratessimilar to checkpoint blockade. In addition, RMC-4550 drovedirect, selective depletion of protumorigenic M2 macrophages viaattenuation of CSF1 receptor signaling and increased M1 macro-phages via a mechanism independent of CD8þ T cells or IFNg .These dramatic shifts in polarized macrophage populations infavor of antitumor immunity were not seen with checkpointblockade. Consistent with a pleiotropic mechanism of action,RMC-4550 in combination with either checkpoint or CSF1R

blockade caused additive antitumor activity with completetumor regressions in some mice; tumors intrinsically sensitiveto SHP2 inhibition or checkpoint blockade were particularlysusceptible. Our preclinical findings demonstrate that SHP2thus plays a multifaceted role in inducing immune suppressionin the tumor microenvironment, through both targeted inhibi-tion of RAS pathway–dependent tumor growth and liberation ofantitumor immune responses. Furthermore, these data suggestthat inhibition of SHP2 is a promising investigational thera-peutic approach.

Significance: Inhibition of SHP2 causes direct and selectivedepletion of protumorigenic M2 macrophages and promotesantitumor immunity, highlighting an investigational therapeuticapproach for some RAS pathway–driven cancers.

IntroductionAllosteric inhibition of the protein tyrosine phosphatase SHP2

(encoded by PTPN11), an established signaling node in the RAS–MAPK growth and survival pathway, is a novel, investigational ther-apeutic strategy for patients bearing tumors with specific oncogenicmutations in this pathway (1–4). SHP2 is a positive transducer ofreceptor tyrosine kinase (RTK) signaling [see Frankson and colleaguesfor a recent (5) review], but themolecularmechanism is still unclear.Weand others have shown that SHP2 acts upstream of RAS and promotesRTK-mediated RAS nucleotide exchange and activation, likely througha scaffolding interaction with SOS1 (1, 6, 7). Clinical studies withinvestigational SHP2 inhibitors are ongoing, and preliminary signs ofclinical activity in patients with non–small cell lung cancer (NSCLC)harboring KRAS mutations, particularly KRASG12C, have beenreported (8). SHP2 is also widely expressed in hematopoietic cells,including both lymphoid and myeloid cells, and there is emergingevidence to support a role in antitumor immunity. The majority ofreported studies have focused on establishing a role for SHP2 in the

regulation of T-cell function (9–11), although recently myeloid-restricted deletion of SHP2 in mice was shown to suppress melanomagrowth (12). Tumor-associated myeloid cell infiltration is associatedwith clinical resistance to immunotherapies (13) and correlates with anegative prognosis for most tumor types (14–22). Identification oftherapeutic strategies that can modulate the recruitment, survival,and/or reprograming of tumor-associated macrophages (TAM) andimprove the clinical response to currently available immunotherapies iscritical (23). Building a comprehensive understanding of the impact, ifany, of allosteric inhibition of SHP2 on innate and adaptive immunity,and how this can influence the clinical response to checkpoint blockade,is fundamental to realizing the full potential of this molecular targetedtherapeutic strategy.

SHP2 may also be an important signaling node downstream ofinhibitory receptors in immune cells. SHP2 binds to tandem phos-phorylated immunoreceptor tyrosine-based inhibition motif (ITIM)and immunoreceptor tyrosine-based switchmotif (ITSM) domains onregulatory receptors in immune cells, including inhibitory immunereceptors like PD-1 and BTLA (24–26), and multiple reports havedemonstrated a SHP2/PD-1 physical interaction in vitro (25, 27–33).Regulation of T-cell receptor signaling in vitro by SHP2 associationwith CTLA4 has also been reported (34), although canonical ITIM/ITSM domains are not present in CTLA4, so the significance of thesereported associations is unclear (35). More recently, through theapplication of cell-free biochemical experiments, it has been proposedthat SHP2 transduces the PD-1–inhibitory checkpoint signal by directdephosphorylation of the costimulatory molecules CD28 and CD226and, consequently, limits T-cell activation (28, 36). Collectively, thesestudies have pointed to a role for SHP2 in regulation of T cells.However, using a T-cell–specific SHP2-deficient mouse model, Rotaand colleagues concluded that SHP2 is dispensable for PD-1 signaling

1Department of Biology, Revolution Medicines, Inc., Redwood City, California.2Department of Chemistry, Revolution Medicines, Inc., Redwood City, California.

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

Corresponding Author: Jacqueline A.M. Smith, Revolution Medicines, 700Saginaw Drive, Redwood City, CA 94063. Phone: 650-481-6920; E-mail:[email protected]

Cancer Res 2020;80:2889–902

doi: 10.1158/0008-5472.CAN-19-3038

�2020 American Association for Cancer Research.

AACRJournals.org | 2889

on October 12, 2020. © 2020 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst April 29, 2020; DOI: 10.1158/0008-5472.CAN-19-3038

http://crossmark.crossref.org/dialog/?doi=10.1158/0008-5472.CAN-19-3038&domain=pdf&date_stamp=2020-6-17

http://crossmark.crossref.org/dialog/?doi=10.1158/0008-5472.CAN-19-3038&domain=pdf&date_stamp=2020-6-17

http://cancerres.aacrjournals.org/

in T cells in vivo, as well as for the global induction of T-cellexhaustion (11), a process that PD-1 has been implicated in control-ling. Furthermore, the control of immunogenic tumors was notimproved in these T-cell SHP2–deficient mice, and the response toanti–PD-1 checkpoint blockade therapy was not affected (11). Oneplausible explanation for the apparent discrepancy between theseobservations is that redundant mechanisms, such as the relatedtyrosine phosphatase SHP1, can mediate PD-1–inhibitory signalingin the setting of SHP2 deficiency (37). The emergence of these types ofcompensatory signaling mechanisms highlights the limitations ofusing genetically engineered mouse models to interrogate thein vivo mechanism(s) of action of SHP2. Moreover, the selectivedeletion of SHP2 protein from only a subset of immune cells obscuresthe clinical implications of the findings thus far, as it does notappropriately model the effects of pharmacologic inhibition of SHP2broadly in multiple immune cell types in addition to tumor cells.

The recent availability of selective, orally-bioavailable small-molecule allosteric inhibitors of SHP2 provides an opportunity tointerrogate the immunomodulatory mechanism(s) of action of SHP2in vivo using pharmacologic tools that circumvent the various limita-tions imposed by genetic approaches. Accordingly, Zhao and collea-gues have reported that a selective, but low potency, small-moleculeinhibitor of SHP2 decreases tumor burden by augmenting cytotoxicT-cell–mediated antitumor immunity (9). However, in this study noevidence was provided to substantiate a direct effect of the SHP2inhibitor on T cells in vivo, and further, the impact of SHP2 inhibitionon the myeloid compartment was not evaluated (9).

In this study, we used the previously described potent and selectiveallosteric inhibitor of SHP2, RMC-4550 (1), to generate an in-depthunderstanding of the integrated effects of SHP2 inhibition in vivo inthe tumor microenvironment. Using syngeneic mouse tumor models,we reveal an unanticipated impact of SHP2 on tumor immunitythroughmodulation of both innate and adaptive immune cells. Similarto immune checkpoint blockade, RMC-4550 caused an increase inCD8þ T-cell tumor infiltrates. RMC-4550 also produced a direct andselective depletion of protumorigenicM2macrophages through atten-uation of CSF1 receptor (CSF1R) signaling. The antitumor effects ofRMC-4550 were additive with either immune checkpoint inhibitors oran anti–CSF1R antibody, consistent with a pleiotropic role for SHP2 inthe tumor microenvironment. Tumors that are intrinsically sensitiveto SHP2 inhibition and also sensitive to checkpoint blockade wereparticularly susceptible to RMC-4550 alone or the combinationtreatment.

Collectively, these findings highlight that SHP2 inhibition is apromising molecular therapeutic strategy in cancer with potentialdual activity: targeted suppression of tumor-intrinsic RAS/MAPK-dependent growth and promotion of antitumor immune responsesthrough transformation of the suppressive tumor immune microen-vironment. Translation of the preclinical combination advantages of aSHP2 inhibitor and checkpoint blockade into the clinical settingwouldbe a significant advance for patients bearing oncogenic RAS pathwayalterations and for whom current therapeutic options and benefits arelimited.

Materials and MethodsCell lines and reagents

All cell lines were obtained from the ATCC except for MC38(NTCCChina). Cells were grown in RPMI (CT26.WT, A20, and4T1) or DMEM (MC38, EMT6, B16-F10) supplemented with 10%heat-inactivated FBS and 1% penicillin/streptomycin (Gibco). Cells

weremaintained at 37�C in a humidified incubator at 5%CO2. All cellswere mycoplasma free, and identity was confirmed by short tandemrepeat profile. Antibodies used for in vivo treatment were fromBioXcell: anti–PD-L1 (10F.9G2), rat IgG2b (LFT-2), anti-PD1(RMP1-14), rat IgG2a (2A3), anti–CTLA4 (9D9), mIgG2b (MPC11);anti–CSF1R (AFS98), rat IgG2a (2A3), anti-CD4 (GK1.5), andanti-CD8 (2.43).

In vivo tumor challengeAll studies were compliant with all relevant ethical regulations

regarding animal research in accordance with approved InstitutionalAnimal Care and Use Committee protocols at MI Bioresearch, Inc.,WuXi Apptec, and HD Biosciences. Female (6 to 8 weeks old)immunocompetent mice were implanted subcutaneously with 5Eþ05CT26.WT cells, 5Eþ05 A20 cells, or 5Eþ05 EMT6 cells (BALB/c,Envigo or BALB/c Rag2 ko/ko, Taconic), and 2Eþ05MC38 or 5Eþ04B16-F10 cells (C57BL/6J, SLAC Laboratory Animal Co., LTD.); orinjected in the mammary fat pad with 5Eþ05 4T1 cells (BALB/c,Shanghai Lingchang Biological Technology Co., LTD.). Once tumorsreached an average size of 48 to 90 mm3, administration of RMC-4550(30 mg/kg, by daily oral administration) or vehicle (2% HPMCin 50 mmol/L sodium citrate buffer), anti–PD-1, anti–PD-L1 oranti–CTLA4 (10 mg/kg, by intraperitoneal administration every3 days), or anti–CSF1R (2 mg per mouse on day 1 followed by 0.2 mgper mouse 16 days after cell implantation, by intraperitoneal admin-istration), was initiated. Experiments with A20 or CT26 where anti–PD-L1 was investigated were not staged, and treatment started 3 daysafter cell implantation. The selective depletion of tissue macrophagesby anti–CSF1R administration was confirmed in liver by flowcytometry.

In vivo immune cell depletion experimentsTreatment began on day 7 at an overall mean tumor burden of

72 mm3. Anti-CD4, anti-CD8, or combination were administeredintraperitoneally (0.5 mg per mouse on days 7, 8, and 9 followed by0.2 mg per mouse on days 13 and 17). RMC-4550 was administeredorally (30 mg/kg daily during 21 days starting at day 9). Depletion ofimmune cells in blood was confirmed by flow cytometry.

Immune phenotyping studies in tumorsTreatment with anti–PD-L1 (10 mg/kg intraperitoneal on days 3, 6,

10, and 13), RMC-4550 (30 mg/kg, by oral daily on days 3 to 15), orcombination started on day 3, and tumors were processed for analysison day 16 after cell implantation. Treatment with anti–CTLA4(10 mg/kg, intraperitoneal on days 7, 10, and 14), RMC-4550(30 mg/kg, daily oral on days 7 to 15), or the combination startedon day 7 (79 mm3 tumors), and tumors were processed for analysis onday 16. Tumors were dissociated into single-cell suspension (Gentle-MACS C tubes and tumor dissociations Kit from Milteny Biotec).Antibodies used included CD3 (145-2C11, Biolegend), CD4 (RM4-5,BD Biosciences), CD8a (53-6.7, BD Biosciences), CD45 (30-F11,Biolegend), CD25 (PC61, Biolegend), PD-1 (29F.1A12, Biolegend),FoxP3 (3G3, ThermoFisher) and MHC Class I (34-1-25, Biolegend),CD11b (M1/70, BD Biosciences), Ly6C (HK1.4, Biolegend), F4/80(BM8, Biolegend), MHC Class II (proprietary from MI Bioresearch),CD45 (30-F11, BDBiosciences), CD206 (CO68C2, Biolegend), CD11c(N418, ThermoFisher), Ly6G (1A8, BD Biosciences), CD19 (1D3, BDBiosciences) and PD-L1 (B7H1, Biolegend), Ki67 (Biolegend, 16A8),and AH1 Dextramer (Immudex JG3294). ACK Lysing Buffer (Bio-legend), Zombie Viability Dye (Biolegend), Fc blocking agent (anti-CD16/32, Biolegend), FoxP3 Fix/Perm kit (eBiosciences), AbC Total

Quintana et al.

Cancer Res; 80(13) July 1, 2020 CANCER RESEARCH2890




compensation (ThermoFisher), and cell staining buffer (BD Bio-sciences) were used. Samples were run in an Attune NxT flowcytometer.

IHC detection for CD8a, F4/80, in mouse paraffin-embeddedtumors

Anti-mouse CD8a (Cell Signaling Technology, 98941, 1.6 mg/mL)or anti-F4/80 (Cell Signaling Technology, 70076, 1.4 mg/mL) rabbitmonoclonal antibodies were used with citrate-based pH 6.2 Heat-Induced Epitope Retrieval. Sections (5mm)were stained on the BiocareintelliPATH platform using the manufacturer's recommended set-tings. Antibody binding was detected with MACH4 HRP-polymerDetection System followed by IntelliPATH FLX DAB chromogen andIntelliPATH Hematoxylin kits. All reagents were from Biocare Med-ical. TissueScope LE whole slide scanner (Huron Digital Pathology),Huron Viewer software, and HALO Image Analysis software fromIndica labs were used for analysis.

PD-1 NFAT Luciferase Reporter AssayEngineered CHO-K1 cells (BPS Bioscience, 60536) were incubated

overnight in RPMI medium supplemented with 10% heat-inactivatedFBS and 1% penicillin/streptomycin (Gibco). Engineered Jurkatcells (BPS Bioscience, 60535) were preincubated with RMC-4550 oranti–PD-1 for 30 minutes and added to CHO-K1 cells. After 16 hours,One-Step Luciferase Assay system (BPS Bioscience, 60690) was addedaccording to manufacturer's instructions, and luminescence was mea-sured on an EnVision Multilabel Plate Reader (Perkin Elmer).

Staphylococcus aureus enterotoxin B superantigen T-cellstimulation assay

Human buffy coat was obtained from San Diego Blood Bank.Peripheral blood mononuclear cells (PBMC) were isolated usingSpMate (Stemcells) and treated with anti–PD-1 (S228P, Invivogen),isotype control (Human IgG4, Invivogen), RMC-4550, or vehicle(0.2% DMSO) at concentrations indicated. Thirty minutes to 1 hourafter compound treatment, cells were stimulated with staphylococcalenterotoxin B (SEB; 0.1 mg/mL, Toxin Technologies), followed byincubation in presence of SEB and compounds at 37�C with 5% CO2

incubator for 3 days. IL2 content was analyzed in supernatant bystandard ELISA (Abcam) with Perkin Elmer Envision MicroplateReader.

Mixed lymphocyte reactionMonocytes were isolated from fresh PBMC from healthy donors

(EasySep monocyte enrichment kit, Stemcell). Monocytes were dif-ferentiated (3 days) and matured (3 days) into monocyte-deriveddendritic cells (Mo-DC) by using Milteny Biotec reagents. Cells wereimmunophenotyped with CD14, CD209, and CD83, and purity wasconfirmed to be >90%. Responder CD3þ T cells were prepared from adifferent donor using a negative selection kit (Stemcell) to obtainuntouched T cells. Cells were cocultured at a final ratio of T cells toMo-DCs of 10:1. Anti–PD-1 (S228P, Invivogen), isotype control(Human IgG4, Invivogen), RMC-4550, or vehicle was incubated for5 days, and supernatants were assessed for IFNg by ELISA (Invitrogen).

In vitro studies with murine bone marrow–derivedmacrophages

Culture of bone marrow–derived macrophages (BMDM): BM wasisolated from the femurs of BALB/c mice. BM cells were plated incomplete Alpha-MEM media (Gibco) containing 10% heat-inactivated FBS (VWR) and 1% Pen-Strep (Corning) and supplemen-

ted with CSF1 (Peprotech) at 10 ng/mL or GMCSF (Peprotech) at25 ng/mL. Growth inhibition and apoptosis assays: After 7 days ofculture, BM cells were dissociated (Gibco Cell Dissociation Buffer)and plated with media containing CSF1 at 10 ng/mL or GMCSF at25 ng/mL. After 3 hours, cells were treated with either RMC-4550 orBLZ-945 (Selleckchem). Cell proliferationwasmeasured 72 hours aftercompound treatment using the CellTiterGlo reagent (Promega). Cas-pase activity was measured using the Caspase-Glo 3/7 kit (Promega)48 hours after compound treatment. Polarization assay: BM cells wereisolated as above and cultured in 10 ng/mL CSF1 for 6 days. Theappropriate cytokine (R&DSystems) was added (M1: IFNg : 20 ng/mL,LPS: 100 ng/mL M2: IL4: 20 ng/mL), and cells were cultured for anadditional 24 hours. Polarized BMDMs were treated with compoundin the presence of CSF1 and the appropriate cytokine and analyzed forcell proliferation and caspase-3/7 activity as described. PhosphorylatedERK (pERK) assay: BM cells were isolated as above and cultured ineither CSF1 or GMCSF for 7 days. Growth factor was removedovernight, and cells were treated with compound for 1 hour.Cells were acutely stimulated with CSF1 at 100 ng/mL or GMCSF at50 ng/mL for 10 minutes. pERK was analyzed using the AlphaLisaSureFire p-ERK1/2 Thr202/Tyr204 kit (PerkinElmer) according to themanufacturer's instructions. pAKT was assessed using the pAKT(Ser473) MSD kit (Meso Scale Diagnostics) according to the manu-facturer's instructions.

Statistical analysisQuantitative data are presented as the mean � SD or the SEM, as

specified in the figure legends. Statistical tests were performed usingGraphPad Prism 7.0. Two-sided Student t tests were used for compar-isons of the means of data between two groups, and one-way ANOVAwith post hoc Tukey test was used for comparisons among multipleindependent groups, unless otherwise specified. For animal studies,animals were randomized before treatments, and all animals treatedwere included for the analyses. P value < 0.05 was consideredsignificant.

ResultsSHP2 inhibition induces antitumor immunity in vivo incheckpoint blockade–sensitive tumors

We first examined the antitumor efficacy of the SHP2 inhibitorRMC-4550 in three syngeneic tumormodels that are partially sensitiveto checkpoint blockade: A20 B-cell lymphoma and both MC38 andCT26 colon carcinomas. RMC-4550 had a modest effect on growth ofA20 cells in 3D in vitro culture but did not reduce the viability ofMC38or CT26 cancer cells at concentrations achievable in vivo (IC50 ¼2 mmol/L, >10 mmol/L, and 10 mmol/L, respectively; SupplementaryFig. S1A; ref. 1). RMC-4550 did inhibit RAS–MAPK signaling, asmeasured bypERK levels, inA20 andMC38 cells (IC50 of 4mmol/L and22 nmol/L, respectively, Supplementary Fig. S1A) but not inCT26 cells(IC50 > 10 mmol/L; Supplementary Fig. S1A). Repeated oral dailydosing of RMC-4550 at 30mg/kg significantly slowed tumor growth ineach of thesemodels (Fig. 1A). No effect of RMC-4550 onCT26 tumorgrowth was observed in vivo when tumors were established in RAG2–deficient mice, which lack T and B lymphocytes and are thus immu-nocompromised (Fig. 1B). These data, togetherwith the lack of in vitroeffect on both viability and RAS–MAPK signaling in CT26 cells(Supplementary Fig. S1A), provided confidence that the observedefficacy in vivo was a function of SHP2-mediated effects on immunecells in the tumor microenvironment. To corroborate these findings,we demonstrated that RMC-4550 did not inhibit tumor growth in

SHP2 in Macrophages and Tumor Immunosuppression

AACRJournals.org Cancer Res; 80(13) July 1, 2020 2891




A

D

B

C CT26, wild-type mice, antibody-mediated immune cell depletion

10 20 30 40 50Days after implant

10 30 50 700

255075

100

Days after implant

%M

ice

with

tum

ors

<2,0

00m

m3

10 20 30 40Days after implant

CT26 colonMC38 colonA20 B cell


CT26, RAG2-/- mice

Vehicle RMC-4550

0

1,000

2,000

10 20 30 400

1,000

2,000

Days after implant10 20 30 40

Days after implant

0

1,000

2,000

Vehicle RMC-4550Tu

mor

vol

ume

(mm

3 )

Isotype control

CD4+/CD8+ T-cell depletion

CD8+T-cell depletion

0255075

100

****

10 20 30 400

255075

100

Days after implant

0255075

100

% M

ice

with

tum

ors <

2,0

00 m

m3

E

* **** ****

10 20 30 40 500

255075

100

Days after implant

%M

ice

with

tum

ors

< 2,0

00m

m3

10 20 30 40 500

1,000

2,000

Days after-implant

Tum

or v

olum

e (m

m3 )

Tum

or v

olum

e (m

m3 )




Isotype RMC-4550Anti−PD-L1 Combination4 TFS

******

CT26 colon

10 20 30 40 500

255075

100

Days after implant

%M

ice

with

tum

ors

<2,

000

mm

3

10 20 30 40 500

1,000

2,000

Days after implant10 20 30 40 50



Days after implant

Isotype RMC-4550Anti–CTLA4 Combination

1 TFS

********

CT26 colon

Figure 1.

SHP2 inhibition induces antitumor immunity in checkpoint blockade–sensitive tumors and is additive in combination with a checkpoint inhibitor. A and B,Oral dailyadministration of 30mg/kgRMC-4550was used in all experiments.A,Activity of RMC-4550 inA20,MC38, or CT26 tumor–bearing immunocompetentmice. Kaplan–Meier plot showing percentage of animalswith tumor burden below 2,000mm3 for the duration of this study.B, Lack of activity of RMC-4550 in CT26 tumor–bearingRAG2–deficient immunocompromised mice. C, Activity of RMC-4550 in CT26 tumor–bearing immunocompetent mice depleted of CD4þ and CD8þ T cells or CD8þ

T cells alone. Left two plots show tumor growth of individual mice for each experimental group described, and right plot shows Kaplan–Meier plot displayingpercentage of animals with tumor burden below 2,000 mm3. D, Activity of RMC-4550, anti–PD-L1 (10 mg/kg, i.p. on days 3, 6, 10, and 13 after implantation), orcombination of both in CT26 tumor–bearing immunocompetent mice. Dashed arrow, last day of treatment of RMC-4550. Tumor growth of individual mice for eachexperimental group described and Kaplan–Meier curves (right) are shown. E, Same as D but using anti–CTLA4 (10 mg/kg, i.p. on days 7, 10, 14, and 17after implantation) as checkpoint inhibitor. Kaplan–Meier curves were compared using the Mantel–Cox log-rank test (A–E, except for A20, used log-rank testfor trend); n ¼ 10 animals per group. � , P < 0.05; �� , P < 0.001; �� , P < 0.0001. TFS, tumor-free survivors.

Quintana et al.





immunocompetent mice when both CD4þ and CD8þ T cells had beenfunctionally depleted in vivowith blocking antibodies (Fig. 1C). CD8þ

T-cell depletion alone completely abrogated the efficacy of RMC-4550,indicating that these immune effector cells are essential for SHP2inhibitor–mediated antitumor immunity (Fig. 1C). Depletion ofCD4þ T cells inhibited tumor growth in vehicle-treated mice, andno further inhibition of growth was apparent with RMC-4550 (Sup-plementary Fig. S1B).

SHP2 inhibition is additive in combination with a checkpointinhibitor

RMC-4550 induced significant tumor growth inhibition of CT26tumors that was superior to anti–PD-L1 (Fig. 1D) and comparableto anti–CTLA4 (Fig. 1E). The combination of RMC-4550 with anti–PD-L1 demonstrated robust antitumor benefit as evidenced by asignificant increase in the time to reach endpoint tumor burden andby tumor regressions in 4 of 10mice (Fig. 1D). In contrast, RMC-4550or anti–PD-L1 treatment alone did not result in any tumor-freeanimals. Tumor-free survivors remained tumor-free for at least 40 daysand, importantly, were resistant to tumor reimplantation, suggestinglong-lasting adaptive immunity (Supplementary Fig. S1C). All treat-ments, including the combination, were well tolerated (SupplementaryFig. S1D). Similar effects were observed with anti–CTLA4, althoughthe combination regimen was less well tolerated (Fig. 1E; Supple-mentary Fig. S1E).

SHP2 inhibition does not confer sensitivity to checkpointblockade in PD-1 refractory models

We tested the hypothesis that combination treatment with a SHP2inhibitor could confer sensitivity to checkpoint blockade using twosyngeneic models that are refractory to anti–PD-1 treatment, 4T1 andB16-F10. 4T1 breast carcinoma is a RAS/MAPK-dependent syngeneicline sensitive to both MEK (trametinib, IC50 3D-growth ¼ 5 nmol/L)and SHP2 (RMC-4550, IC50 3D-growth¼ 33 nmol/L, SupplementaryFig S1A) inhibition in vitro, whereas B16-F10 melanoma cells areinsensitive to SHP2 inhibition in vitro (Supplementary Fig. S1A).RMC-4550 did not increase sensitivity to anti–PD-1 in vivo in either ofthese models, irrespective of whether the cells were sensitive (4T1,Supplementary Fig. S1F) or insensitive (B16-F10, SupplementaryFig. S1G) to the tumor-intrinsic effects of SHP2 inhibition onRAS/MAPK signaling.

SHP2 inhibition stimulates adaptive immunityAnalysis of the immune landscape of CT26 tumors demonstrated

that RMC-4550 treatment increased the percentage of CD3þ T cells by2-fold, from a baseline of 8% � 3% of CD45þ tumor-infiltratingleukocytes (TIL; Supplementary Fig. S2A and S2B). CD8þ T-cellfrequency was increased in tumors from RMC-4550–treated mice,whereas there was no change in CD4þ T or T regulatory (Treg) cellfrequency (Fig. 2A and B). Furthermore, the CD8þ T cells expressedless of the inhibitory molecule PD-1 (Fig. 2B). The increase in CD8þ

T-cell frequency was comparable with that observed with checkpointblockade, and the combination of RMC-4550 with either anti–PD-L1or anti–CTLA4 exhibited additivity (Fig. 2A and B). The combina-torial effect with anti–CTLA4 on CD8þ T-cell frequency was statis-tically significant (Fig. 2B). RMC-4550 and anti–CTLA4 treatment–evoked increases in CD8þT cells weremostly localized to the border ofthe tumor, and although an increase relative to vehicle control wasapparent for each of the single-agent treatment regimens, only in thecase of the combination of RMC-4550 with checkpoint blockade didthe increase reach statistical significance (Fig. 2C).

Following RMC-4550 treatment, a higher frequency of the CD8þ

tumor-infiltrating T cells were specific for the tumor-associated anti-gen AH1 (analyzed by dextramer staining; Supplementary Fig. S2C)and exhibited an activated profile, as they were proliferative (Ki67staining) and expressed the cytotoxic cytokine IFNg (intracellularstaining; Supplementary Fig. S2C). These changes did not reachstatistical significance but collectively are consistent with functionalactivation of the CD8þ tumor-infiltrating T cells.

RMC-4550 also increased the expression of class I MHC moleculesand PD-L1 in CD45-negative tumor cells, similarly to anti–CTLA4(Fig. 2D). These effects were dependent on IFNg and CD8þ T cells, asthey were abrogated by depletion with the corresponding antibodiesin vivo (Fig. 2D). Consistent with the lack of intrinsic effects ofRMC-4550 on proliferation of CT26 cells in vitro, the proliferationofCT26 cells in vivowasnot affected, asmeasured byKi67 staining (FigS2D). Finally, consistent with previous report (38), SHP2 inhibitiondid not affect CT26 tumor vascularization as analyzed by CD31 tissuestaining (Supplementary Fig. S2E).

SHP2 inhibition does not phenocopy the effect of anti–PD-1in T cells

The direct effects, if any, of SHP2 inhibition onT cells were exploredin vitro and in vivo and were compared with those of immunecheckpoint blockade. Focusing on the proposed role of SHP2 as adownstream transducer of PD-L1/PD-1 signaling (25, 28), we obtainedrobust biochemical evidence that the tandem phosphorylated ITIMand ITSM in PD-1 can activate the autoinhibited form of SHP2.Titration of purified full-length SHP2 with a synthetic peptide thatmimics the PD-1 tandem phosphorylated ITIM/ITSM increasedenzyme activity by 270-fold (EC50 of 3.2 nmol/L; SupplementaryFig. S2F). The PD-1 peptide (10 nmol/L) induced activation of theautoinhibited form of SHP2was blocked by RMC-4550 with an IC50 of7.1 nmol/L. To monitor PD-L1/PD-1 signal transduction in a cellularcontext, we used a bioluminescent reporter assay in Jurkat T cells.These Jurkat cells were engineered to express human PD-1 and aluciferase reporter driven by an NFAT response element, and werecocultured with a variant of CHO cells that can serve as antigen-presenting cells (APC). These APCs are CHO-K1 cells expressinghuman PD-L1 and an engineered cell surface protein designed toactivate cognate T-cell receptors in an antigen-independent manner.RMC-4550 caused a concentration-dependent activation of the NFATluciferase reporter with an apparent potency (EC50 ¼ 3.5 nmol/L)consistent with an on-mechanism effect for the SHP2 inhibitor.However, the maximal signal induction was approximately 4-foldlower than that observed with anti–PD-1 (Fig. 2E). In human PBMCcultures, both RMC-4550 and anti–PD-1 enhanced IL2 secretion inresponse to the superantigen SEB, but the response to RMC-4550 wasnot prominent compared with that of anti–PD-1 (Fig. 2F, top).Furthermore, RMC-4550 (up to a test concentration of 5 mmol/L)did not elicit a response in human T cells during a mixed lymphocytereaction, whereas anti–PD-1 produced a robust increase in IFNgrelease (Fig. 2F, bottom).

Given the equivocal findings in vitro, we elected to use an in vivomodel to investigate the role of SHP2 in PD-1 signaling. Checkpointblockade has been shown to reduce CD8þ T-cell exhaustion indifferent systems including the lymphocytic choriomeningitis virus(LCMV) infectionmousemodel (39). A role forCD28 costimulation inCD8þ T-cell rescue in the LCMVmodel has been confirmed (40), andwe used this model to determine whether SHP2 inhibition mimics theeffects of anti–PD-1 on T-cell exhaustion and viral load reductionsin vivo. In our study, mice were challenged with LCMV clone 13 to






A

Isotype

RMC-4550Anti–CTLA4

Combination05

101520

CD

8+ T

(%C

D45

+ ) ****

*

*

*

0

20

40

60

CD

8+ T/

Treg

0

2

4

6

CD

4+ T

(%C

D45

+ )

02468

10P

D-1

ofC

D8+ T

(MFI

,x10

4 )

*****

********

B

Isotype

RMC-4550Anti–PD-L1

Combination012345

CD

4+ T

(%C

D45

)

05

101520

CD

8+ T/

Treg

0

5

10

15

CD

8+ T

(%C

D45

)

02468

10

PD

-1of

CD

8+T

(MFI

,×10

4 )

D

02468

MH

CI o

fC

D45

-neg

ativ

ece

lls (M

FI, ×

104 )

α-IFNγ α-CD8+Isotype

******

1.0

1.2

1.4

1.6

PD

-L1

ofC

D45

-neg

ativ

ece

lls (M

FI, ×

103 )

******


Vehicle RMC-4550Anti–CTLA4

C Vehicle

RMC-4550

An�–CTLA4

Combina�on

E

−9 −8 −7 −6 −502468

log [Anti-PD–1 (g/mL)]

NFA

TLu

cife

rase

(AU

×104 )

−10 −9 −8 −7 −6log [RMC-4550 (mol/L)]

IFN

γ(p

g/m

L)

0

75

150

225

IL2

(pg/

mL)

0

500

1,000

RMC-4550Anti–PD-1

F

Vehicle


Combination

05

10152025 *

Edgetumor area

0

5

10

15

CD

8+ T

posi

tive

cells

(%) *

Totaltumor area

Figure 2.

SHP2 inhibition stimulates adaptive immunity in similarmanner to checkpoint inhibition. CT26 tumors derived from similar experiments as shown in Fig. 1D or Eweretaken at day 16, after 13 or 9 days of treatment with anti–PD-L1 (A) or anti–CTLA4 (B–D), respectively, and tumor immune cell infiltrates were analyzed by flowcytometry or IHC. A and B, Quantification by flow cytometry of CD8þ T or CD4þ T cells in CD45þ TILs, ratio of CD8þ T/Treg, and PD-1 mean fluorescence intensity(MFI) of CD8þT cells.C,Quantification of CD8þT-positive cells in CT26 tumors, as percentage of total number of cells in each tumor section (middle graph) or in edgeof tumor (right), determinedby IHC.D,Quantification byflowcytometry ofMHCclass I or PD-L1MFI of CD45-negative tumor cells and after in vivodepletion of IFNg orCD8þTcells.E,NFAT luciferase reporter genePD-1/PD-L1 bioassay showsNFATactivation in response to increasing concentrations of anti–PD-1 or RMC-4550.F,SEB(top) or mixed lymphocyte reaction (bottom) assay with human cells from healthy donors shows secretion of IL2 or IFNg in response to increasing concentrations ofanti–PD-1 (1 ng/mL to 10mg/mL) or RMC-4550 (0.1 nmol/L to 5mmol/L).A–C,Data represent analysis of 5mice per group,mean�SEM.One-wayANOVA followedbyHolm–Sidak test (A and B) or Tukey (C and D). � , P < 0.05; �� , P < 0.01; �� , P < 0.001; �� , P < 0.0001.

Quintana et al.





establish a chronic infection, followed by administration of RMC-4550or anti–PD-L1. RMC-4550 induced a significant increase in thefrequency of CD8þ T cells in the spleen, but this was not accompaniedby a significant increase in antigen-specific CD8þ T cells. Ultimately,RMC-4550 failed to decrease viral titers in peripheral organs (Sup-plementary Fig. S2G). In contrast, anti–PD-L1 treatment did effec-tively increase antigenic CD8þ T cells, correlating with higher viralcontrol in various organs (Supplementary Fig. S2G).

In summary, although we cannot rule out a role for SHP2downstream of PD-1 signaling, we have demonstrated, usingvarious model systems in vitro and in vivo, that SHP2 inhibitionand PD-1 blockade are not equivalent with respect to directmodulation of T-cell function. Rather, it seems likely that SHP2inhibition can restrain PD-1 signaling to some extent, but that thefull downstream effects are blunted, potentially due to the recruit-ment of redundant signaling effector molecules.

SHP2 inhibition modulates innate immunity, an effect not seenwith checkpoint blockade

To explore additional mechanisms of SHP2 inhibitor actionin vivo beyond transduction of checkpoint signals, we focused onmyeloid cells in the tumor microenvironment. CT26 tumors arerich in myeloid cells; CD11bþ cells constitute 79%�2% of CD45þ

TILs and 64% � 2% of those are F4/80þ TAMs. RMC-4550treatment had a striking impact on tumor myeloid infiltrates, inparticular macrophages, inducing a 3-fold decrease in the frequencyof F4/80þ cells among CD45þ TILs (Fig. 3A–C; SupplementaryFig. S3A). This finding was confirmed by IHC staining (Fig. 3D);the decrease in macrophages was most evident in the core of thetumor (Fig. 3D; Supplementary Fig. S3B).

TAMs are highly plastic and can acquire different phenotypes in thetumor microenvironment ranging from proinflammatory M1 TAMs(MHCIIhigh and CD206negative/low) to protumorigenic M2 TAMs(MHCIIlow and CD206high; ref. 41). RMC-4550 induced a significantdecrease in the frequency of M2, the predominant population in CT26tumors (>90% of TAMs, Fig. 3A–C), and an increase in the frequencyof M1 among CD45þ TILs; by extension, the M2/M1 ratio wasdramatically reduced (Fig. 3B and C). Checkpoint blockade elicitedonly a modest effect on TAM frequencies (Fig. 3B and C), but thecombination of checkpoint blockade and RMC-4550 drove an evendeeper modulation of TAMs (Fig. 3B and C). Checkpoint blockadepreviously has been shown to modulate TAM frequencies indirectly,via modulation of CD8þ T-cell frequency and IFNg secretion in thetumor microenvironment (42, 43). In contrast, RMC-4550–mediatedmodulation of M2-TAM frequencies was unchanged by depletion ofeffector cells or IFNg cytokine (Fig. 3E). As expected, IFNg or CD8þ

T-cell depletion decreased the overall frequency of M1-TAMs; how-ever, a significant RMC-4550–mediated increase was still apparent(Fig. 3E). The expression of MHCI in M1 and M2-TAM was signif-icantly increased with RMC-4550 treatment, and this effect wasdependent of IFNg and CD8þ T cells (Fig. 3F). The expressionof PD-L1 in tumor-associated macrophages was not changed byRMC-4550 treatment (Supplementary Fig. S3E).

Granulocytic myeloid-derived suppressor cells (gMDSC) andmonocytic MDSC (mMDSC) accounted for 7% � 1% and 14% � 1%of CD45þTILs, respectively. Treatment with RMC-4550 increased thefrequency of mMDSCs but had no effect on gMDSCs (Fig. 3G andH).The expression of MHCI or PD-L1 in MDSC was not changed uponRMC-4550 treatment (Supplementary Fig. S3E). To explore potentialfunctional consequences of a SHP2 inhibitor–mediated increase inmMDSC, we used an in vitro suppression assay. Coculture of human

MDSC with T cells induced suppression of T-cell proliferation andIFNg release (Supplementary Fig. S3F). RMC-4550 alone had no effecton T-cell proliferation or cytokine release (Supplementary Fig. S3F)but was able to block the antiproliferative effects of MDSCs on CD8þ

T cells (Supplementary Fig. S3F). A concomitant concentration-dependent increase in IFNg release was also observed (SupplementaryFig. S3F). The viability of MDSCs in vitro was not affected byRMC-4550 (92.5%–93.5% viable compared with 93.3% viable inDMSO-treated MDSCs, determined by flow cytometry).

The frequency of myeloid cells in spleen or peripheral blood oftumor-bearing mice was unchanged with RMC-4550 treatment, sug-gesting that myelopoiesis was not affected at this timepoint (Supple-mentary Fig. S3C and S3D).

In summary, SHP2 inhibition produces a marked shift in polarizedmacrophage populations in the tumor microenvironment in favor ofantitumor immunity, an effect that was not observed upon checkpointblockade. This selective effect of RMC-4550 on myeloid cells mayunderlie the combination benefit of a SHP2 inhibitor and checkpointblockade on tumor growth inhibition (Fig. 1D and E).

SHP2 inhibition suppresses CSF1R signaling and selectivelyaffects viability of M2 macrophages

The prominent reduction in macrophage frequency observedin vivo following administration of RMC-4550, and the apparent lackof dependence on effector lymphocytes or cytokines, is consistent witha direct effect of SHP2 inhibition onmacrophage viability. To evaluatethis possibility, BM cells from BALB/c mice were differentiated withCSF1 or GMCSF in vitro. CSF1 differentiated BMDMs represent apopulation of F4/80þ, MHCIIlow, CD11c- macrophages, whereasGMCSF–differentiated BM cells are MHCIIhigh, CD11cþ, and likelyrepresent a mixture of macrophages and DCs (44). RMC-4550 potent-ly inhibited the growth of CSF1–differentiated (IC50¼ 13 nmol/L) butnot GMCSF–differentiated (IC50 > 1 mmol/L) BM cells (Fig. 4A). Inaddition, SHP2 inhibition selectively induced caspase-3/7 activation,as a marker of apoptosis, in CSF1–differentiated BMDMs (EC50 ¼2.8 nmol/L, Fig. 4B).

The CSF1R is an RTK that controls the survival and proliferationof macrophages (45) and is the target of several therapeutic agents inclinical development for cancer (46). The selective CSF1R kinaseinhibitor BLZ945 (47) also showed selective growth inhibition andinduction of apoptosis in CSF1–differentiated, but not GMCSF–differentiated, BMDMs (Supplementary Fig. S4A and S4B). The timecourse of growth inhibition by BLZ945 or RMC-4550 was similar,and comparable to that caused by CSF1 deprivation (SupplementaryFig. S4C). Given these observations, together with the well-established role of SHP2 as a positive signal transducer downstreamof many RTKs, we hypothesized that SHP2 inhibition suppressesCSF1R signaling. Indeed, we observed strong inhibition of ERK 1/2phosphorylation by RMC-4550 after acute stimulation of BMDMswith CSF1 (IC50 ¼ 3 nmol/L, Fig. 4C). These results were recapit-ulated using a recombinant cell line that reports on CSF1R activationand signaling (Supplementary Fig. S4D). SHP2 inhibition alsodecreased GMCSF–induced ERK 1/2 phosphorylation, albeit to alesser extent (IC50 ¼ 93 nmol/L, Fig. 4C), an effect which was notobserved with BLZ-945 (Supplementary Fig. S4E) and likelyaccounts for the moderate growth-inhibitory effect of RMC-4550in these cells. Importantly, these in vitro results were recapitulated inmonocytes purified from human PBMCs, with SHP2 inhibitionresulting in decreased ERK 1/2 phosphorylation and potent inhibi-tion of growth (IC50¼ 35 nmol/L; Fig. 4D; Supplementary Fig. S4F).Moderate suppression of AKT phosphorylation, another important






A

D

C

0

20

40

60

F4/8

0+(%

CD

45+ )

*******

****

**

0

5

10

M1

(%C

D45

+) ****

****

****

*

0

20

40

60

M2

(%C

D45

+)

********

*******

***

01020304050

M2/

M1

***********

****

Isotype


Combination

0

20

40

60

F4/8

0(%

CD

4 5) **

*****

0

5

10

15

M1

(%C

D45

)

******

0

20

40

60

M2

(%C

D45

) *****

****

0

25

50

M2/

M1

****

***

60708090

100

CD

11b

(%C

D45

)

**

Isotype


Combination

B

Vehicle RMC-4550An�–CTLA4 Combina�on

010203040

F4/8

0P

ositi

vear

ea(%

)

****

*

*

VehicleRMC-4550

E FVehicleRMC-4550

0.0

0.5

1.0

1.5

M1

(%C

D45

) * ***

0

20

40

60

M2

(%C

D45

)


*** *** ***

0

1

2

MH

CI o

f M1+

(MFI

, ×10

4 )

*

012345

MH

CI o

f M2+

(MFI

, ×10

4 )

*


HG

010203040

mM

DS

C (%

CD

45) *

******

*

05

10152025

gMD

SC

(%C

D45

)

010203040

mM

DS

C(%

CD

45+ )

*******

****

**

0

10

20

30

gMD

SC

(%C

D45

+ )Isotype


Combination

Isotype


Combination

CD206

MH

C II

Isotype Anti–CTLA4

CombinationRMC-4550

Figure 3.

SHP2 inhibition modulates innate immunity, an effect not seen with checkpoint blockade. CT26 tumors derived from similar experiments as shown in Fig. 1D and Ewere taken at day 16, after 13 or 9 days of treatment with anti–PD-L1 (B and G) or anti–CTLA4 (A, C, D, and H), respectively, and tumor myeloid cell infiltrates wereanalyzed by flow cytometry or IHC. A, Representative flow cytometric analysis of MHC class II and CD206 expression of TAMs defined as CD45þ/CD3–/CD19–/CD11bþ/Ly6G–/Ly6C low/F4/80þ. Gates indicate strategy to define M1 and M2 TAMs. B and C, Quantification by flow cytometry of CD11bþ, F4/80þ, M1, or M2of CD45þ TILs in each experimental group as indicated. D, IHC analysis for F4/80þ cells in consecutive sections of same tumors analyzed in Fig. 2C. Quantificationof F4/80þ area, as percentage of total area in each tumor section (core and border), is shown on right. E,Analysis by flow cytometry of the frequency of M1þ (top) orM2þ (bottom) TAMs upon in vivo depletion of IFNg or CD8þ T cells in CT26 tumors derived from the same experiment as in Fig. 2D. F, MHCI mean of fluorescenceintensity (MFI) analyzed by flow cytometry on M1 and M2 cells gated as in E. G and H, Quantification by flow cytometry of mMDSC (CD45þ/CD3–/CD19–/CD11bþ/Ly-6CHigh/Ly-6G-) or gMDSC (CD45þ/CD3–/CD19–/CD11bþ/Ly-6Gþ).B–H,Data represent analysis of 5mice per group,mean� SEM. One-wayANOVA followed byTukey. � , P < 0.05; �� , P < 0.01; �� , P < 0.001; �� , P < 0.0001.

Quintana et al.





Figure 4.

SHP2 inhibition suppresses CSF1R signaling and selectively affects viability of M2macrophages. SHP2 inhibition exhibits greater antitumor activity relative to CSF1Rinhibition in vivo.A, The effect of RMC-4550 (72 hours) on proliferation ofmurine BM cells grown in either CSF1 (IC50¼ 13 nmol/L) or GMCSF (IC50 not calculated dueto shallow depth of inhibition). B, The effect of RMC-4550 (78 hours) on caspase-3/7 activation of murine BM cells. C, Effects of RMC-4550 on cellular pERK levelsafter acute stimulation of murine BM cells with CSF1 (IC50¼ 3 nmol/L) or GMCSF (IC50¼ 93 nmol/L).D,Western blot showing effects of RMC-4550 and BLZ-945 onpERK levels of humanmonocytes acutely stimulatedwith CSF1 for 5minutes. E,Murine BMDMswere polarized toM1 (IFNg , LPS) or M2 (IL4) phenotypes for 24 hours.Graph shows growth after treatmentwith RMC-4550 (72 hours).F,M1- andM2-polarizedBMDMswere treatedwith RMC-4550 (48 hours) and assayed for caspase-3/7 activation. A–F, Data represent the mean� SD of n¼ 2 or 3 independent biological experiments performed in technical duplicate. G and H, Activity of RMC-4550(oral daily administration of 30 mg/kg), anti–CSF1R (2 mg/mouse on staging day, followed by 0.2 mg/mouse 6 days after staging), or combination of both in CT26tumor–bearing immunocompetent mice. Tumor growth of individual mice for each experimental group described (G) and Kaplan–Meier curves (H). Kaplan–Meiercurves were compared using the Mantel–Cox log-rank test; n ¼ 10 animals per group. � , P < 0.05; �� , P < 0.01; �� , P < 0.001; �� , P < 0.0001.






signaling node for survival downstream of CSF1R, was observed withRMC-4550 in human monocytes but not in murine BMDMs (Sup-plementary Fig. S4G–S4I).

BMDMs were polarized to either an M1 (IFNg , LPS) or M2 (IL4)phenotype to explore the contribution of a selective intrinsic effect ofSHP2 inhibition on M2 macrophages over M1 in vitro. The M1-polarized macrophages expressed higher levels of inducible nitricoxide synthase, whereas M2 polarization resulted in increased levelsof CD206 and arginase (Supplementary Fig. S4J). M2 macrophageviability was sensitive to RMC-4550 (IC50 ¼ 19 nmol/L), but M1-polarized macrophages remained almost entirely refractory to drugtreatment (IC50 > 1 mmol/L; Fig. 4E). Similarly, SHP2 inhibitionselectively induced caspase-3/7 activation in M2 but not M1 macro-phages (Fig. 4F), which likely accounts for the dramatic decrease inM2frequency observed in vivo (Fig. 3B).

We were unable to determine the impact of SHP2 inhibition onmacrophage differentiation per se because RMC-4550 produced asignificant decrease in monocyte viability when present during thedifferentiation, precluding robust phenotypic characterization of thedifferentiated cells.

Recent data have suggested that increased levels of IFNg and TNFain the tumormicroenvironment, caused by infiltration ofCD8þT cells,can trigger an adaptive response of CSF1 production by certain cancercells (48). This in turn can promote recruitment and proliferation ofimmunosuppressive TAMs, hampering the antitumor immuneresponse to checkpoint inhibitors. Treatment of CT26 cells in vitrowith IFNg and TNFa did increase production of CSF1 mRNA

(Supplementary Fig. S5A). However, we propose that the ability ofRMC-4550 to inhibit CSF1R signaling and decrease immunosuppres-sive TAM populations, as shown herein, would negate any inhibitoryeffects of CSF1 release by tumor cells.

SHP2 inhibition exhibits greater antitumor activity relative toCSF1R inhibition in vivo

The contribution of SHP2-mediated blockade of the CSF1R sig-naling pathway to the antitumor efficacy of RMC-4550 in the CT26model was examined by comparing the response with that of CSF1Rblockade. Anti–CSF1R treatment, in contrast to RMC-4550, did notinduce any significant tumor growth delay (Fig. 4G and H). Thesefindings provide evidence that the in vivo antitumor immunomodu-latory effects of a SHP2 inhibitor reflect more than modulation of themyeloid compartment alone.

The combination of anti–CSF1R and RMC-4550 showed additiveantitumor effects in the CT26 model (Fig. 4G and H). Althoughunexpected, this result may reflect the differential mechanisms ofinhibition of CSF1R signaling by these two agents. Activation ofparallel signaling pathways downstream of CSF1R (e.g., PI3K/AKT)is insensitive to SHP2 blockade (Supplementary Fig. S4I), whereasdirect receptor inhibition likely suppresses additional prosurvivalsignaling pathways. Given the role of both SHP2 and CSF1R as keysignaling nodes in multiple cell types and the complexity of thetumor microenvironment in vivo, further studies are required toelucidate the precise mechanism(s) underlying this combinatorialeffect.

A B

25 50 750

255075

100

Days after implant

% M

ice

with

tum

ors

< 2,

000

mm

30

1,000

2,000

7 14 21 280

1,000

2,000

7 14 21 28

1 TFSAnti–PD-1

Isotype RMC-4550

Combination2 TFS

Tum

or v

olum

e (m

m3 )

Days after implant

RMC-4550 Anti–PD-1 Isotype control

Combination

********

***

EMT6 breast EMT6 breast

C

CD8+T

Figure 5.

SHP2 inhibition is additive in combinationwith checkpoint inhibition in a SHP2inhibitor–sensitive tumor model. A and B,Activity of RMC-4550 (oral daily administra-tion of 30 mg/kg), anti–PD-1 (10 mg/kg, i.p.every 3 days), or combination of both inEMT6 tumor–bearing immunocompetentmice. Tumor growth of individual mice foreach experimental group described (A) andKaplan–Meier curves (B). Kaplan–Meiercurves were compared using the Mantel–Cox log-rank test; n ¼ 10 animals per group.� ,P <0.05; ��,P <0.01; �� ,P <0.001; �� ,P <0.0001. TFS, tumor-free survivors. C, Work-ing model for the effects of SHP2 inhibitionon antitumor immunity via modulation ofboth adaptive and innate mechanisms:blockade of inhibitory signaling in CD8þ Tcells; direct and selective depletion of M2protumorigenic macrophages throughattenuation of CSF1R signaling and inductionof apoptotic cell death; decrease of the sup-pressive potential of mMDSC; and increaseof M1 macrophages via a mechanism that isindependent of CD8þ T or IFNg . Collectively,these mechanisms contribute to generate aless immunosuppressive environment andone that favors tumor cell elimination. Inthose cancers with aberrant RAS/MAPKsignaling, which are intrinsically sensitive toSHP2 inhibition, the ultimate impact ontumor cell growth will reflect integration ofthe both the direct, targeted, and antitumorimmunity mechanisms.

Quintana et al.





SHP2 inhibition is additive in combination with checkpointblockade in a SHP2 inhibitor–sensitive syngeneic model

The combined tumor-intrinsic and immune-mediated antitumoreffects of SHP2 inhibition have not been reported. EMT6 breastcarcinoma is a RAS/MAPK-dependent syngeneic line sensitive toboth MEK (trametinib, IC50 3D-growth ¼ 47 nmol/L) and SHP2(RMC-4550, IC50 3D-growth¼ 100 nmol/L, Supplementary Fig. S1A)inhibition in vitro. RMC-4550 alone significantly inhibited growth ofestablished EMT6 tumors in immunocompetent mice in vivo, aneffect superior to that of anti–PD-1 (Fig. 5A). The combination ofRMC-4550 and anti–PD-1 resulted in sustained tumor growth inhi-bition that greatly increased the time to reach endpoint (Fig. 5B). Thistreatment also led to tumor regressions in 20% of mice, which wereresistant to tumor reimplantation, suggestive of long-lasting adaptiveimmunity (Supplementary Fig. S5B). Treatment of EMT6 tumor–bearing mice with RMC-4550 also induced a significant reduction intumor cell proliferation, asmeasured by Ki67 staining, analyzed 9 daysafter treatment (Supplementary Fig. S5C). These data corroborate thefindings of cell-intrinsic effects of RMC-4550 onproliferation of EMT6cells in vitro (Supplementary Fig. S1A).

Based on the collective observations presented here, we propose amodel inwhich the pleiotropic effects of SHP2 inhibition onboth innateand adaptive immunity cooperate to enhance tumor cell elimination(Fig. 5C). This study reveals a direct role for SHP2 in supporting animmunosuppressive tumormicroenvironment in addition to an impactonproinflammatorymacrophages, although themechanismunderlyingthe effect on M1 macrophages is unclear. We have demonstrated thatCD8þ T cells are obligatory for the antitumor activity of SHP2 inhi-bition; however, the underlyingmechanistic driver(s) of the augmentedadaptive immune response remains to be determined.

DiscussionIn the present study, we demonstrate that SHP2 inhibition pro-

motes antitumor immunity by modulating both innate and adaptiveimmune cells. We propose that, although induction of antitumorimmunity by SHP2 inhibition is T-cell–dependent, a major driver ofthe response is modulation of the macrophage compartment ratherthan a direct effect on T-cell signaling, thus differentiating SHP2inhibition from checkpoint blockade. Our data support a model inwhich SHP2 inhibition has a direct impact on the viability of TAMs,thereby promoting a less immunosuppressive tumor microenviron-ment. An appreciation of the tumor-extrinsic immune-modulatorymechanisms of SHP2 should be instructive to the clinical evaluation ofSHP2 allosteric inhibitors as a novel molecular therapeutic strategy inpatients with cancer.

Consistent with the proposed role of SHP2 as a downstreamtransducer of PD-1 checkpoint signaling in T cells (25, 27–33, 36, 49),we have observed similarities between the in vivo responses to SHP2inhibition and immune checkpoint blockade in the tumor immunemicroenvironment. We and others (9) have shown that an increase intumor-infiltrating CD8þ T cells is essential for SHP2 inhibitor–mediated control of established tumor growth and that these T cellsexpress less PD-1, suggesting that they are less exhausted in response tochronic antigen exposure. However, although we have found a generalconcordance between the responses to anti–PD-1 and SHP2 inhibitionin various in vitro readouts of T-cell function, we have been unable todemonstrate that pharmacologic inhibition of SHP2 is equivalent toPD-1 blockade. In particular, the disparate magnitude of the responsessuggests that SHP2 is not the sole effector of inhibitory PD-1 signalingin these model systems, as has been proposed previously (11, 37). The

failure of RMC-4550 to phenocopy the effects of anti–PD-1 in theLCMV T-cell exhaustion model in vivo also points to a greatercomplexity in PD-1 signaling than has perhaps been appreciated thusfar. In summary, although the present observations are consistent witha role for SHP2 in PD-1 signal transduction and T-cell biology, theprecise role for SHP2 in this pathway vis a vis other redundantmechanisms has yet to be elucidated.

More striking is the enhancement of tumor growth inhibition thatwe and others (9) observe with the combination of global SHP2inhibition and checkpoint blockade; this is indicative of additionalfunctions for SHP2 beyond checkpoint transduction in T cells. Sig-nificantly, we found that SHP2 inhibition had a profound impact onthe survival and function of suppressive monocytic immune cells suchas TAMs and MDSCs. Here, we demonstrate using a pharmacologicapproach that SHP2 is a positive regulatory of ERK signaling down-stream of CSF1R in human monocytes and murine BMDMs, which isin agreement with previous studies using genetic deletion ofPTPN11 (50). The inhibition of CSF1R prosurvival signaling likelyaccounts for the selective effects on M2 macrophage populations, ashas been observed previously with CSF1R inhibitors (51–54), and issupported by the in vitro experiments in the present study. Theselective depletion of M2 macrophages in the tumor microenviron-ment after SHP2 inhibition, without major effects on the M1 popu-lation, has important translational implications. We did not observeeffects of SHP2 inhibition on GMCSF–differentiated macrophagesin vitro, suggesting that SHP2 does not play a role downstream of thisreceptor. The GMCSF receptor transduces prosurvival signals in M1macrophages, which may be an explanation of why SHP2 inhibitionsparesM1 cells and instead has a selective effect onM2s. In addition toits role as a positive regulator of the RAS pathway, SHP2 has also beenproposed to negatively regulate STAT1 activation downstreamof IFNgsignaling (55–57). As IFNg/STAT1 signaling is important in M1macrophage activation (58), inhibition of SHP2 may be supportinga feed-forward loop for M1 macrophage polarization and survival,which encompasses not only macrophage-intrinsic effects on signal-ing, but is influenced by the infiltration of IFNg-producing CD8þ

T cells into the tumor. Consistent with this hypothesis, IFNg or CD8þ

T-cell depletion induced an overall decrease inM1 frequency, althougha SHP2 inhibitor–mediated increasewas still apparent. A role for SHP2downstream of PD-1 in myeloid cells may also be possible. PD-1signaling in myeloid cells can dampen antitumor immunity by reg-ulating lineage fate commitment and function of myeloid cells (59).Myeloid-specific deletion of PD-1 in tumor-bearing mice resulted in adiminished accumulation of immature immunosuppressive cells andan enhanced output of differentiated, inflammatory effectormMDSCs,and phagocytic macrophages, a phenotype similar to that of SHP2inhibition.

Adaptive responses to signals in the tumor microenvironment arenot restricted to the immune compartment. There is compellingevidence that the infiltration of CD8þ T cells can induce productionof CSF1 bymelanoma cells and other cancers by secretion of IFNg andTNF-a (48, 60), an effect we also observed in vitro in the colon CT26model. Increased levels of CSF1 promote an increase in immunosup-pressive M2 macrophages, via CSF1R activation, and a negativecorrelation with overall patient survival (48). The opposing effects ofCD8þ T-cell infiltration induced by checkpoint blockade could becounteracted by combination with anti–CSF1R therapies in a murinemelanoma model (48). Intriguingly, our results suggest that SHP2inhibition has the potential both to induceCD8þT-cell infiltration andsimultaneously to counteract its negative consequences by suppressingCSF1R signaling and therefore contract the immunosuppressive






macrophage population in the tumor microenvironment. This mech-anism of action may contribute to the enhanced antitumor activity weobserved with RMC-4550 in combination with checkpoint blockade.Correspondingly, it may account for the superior tumor growthinhibition observed with the SHP2 inhibitor relative to anti–CSF1R.

The potential for SHP2 inhibitors to provide therapeutic benefit insolid tumors bearing SHP2-sensitive oncoproteins, in particular inNSCLC, is the focus of intensive clinical investigation.Multiple, rationalcombination strategies for a SHP2 inhibitor with agents that targetalternate nodes in the RAS–MAPK pathway [e.g., MEK (6),KRASG12C (61), or RTK (62) inhibitors] or extraproliferative functionsof RAS (e.g., CDK4/6; ref. 63) have also been proposed. The present dataprovide a strong rationale for a clinical combination strategy with aSHP2 inhibitor and agents that target the immune system directly, suchas anti–PD-1 and anti–CSF1R. Patients bearing tumors that harboroncogenic driver mutations sensitive to SHP2, and with establishedclinical sensitivity to checkpoint inhibitors, for example KRASG12C-mutant NSCLC patients, could be particularly susceptible to this com-bination therapy. On the other hand, the present preclinical findingssuggest that SHP2 inhibition seems unlikely to increase sensitivity to animmune checkpoint inhibitor in checkpoint resistant tumors.

In summary, we have shown using preclinical models that SHP2plays a central role in inducing immune suppression in the tumormicroenvironment both by inhibiting T cells and supporting theviability of protumorigenic macrophages. SHP2 inhibition is anattractive investigational therapeutic strategy with potential dualactivity: targeted inhibition of RAS–MAPK-dependent tumor growthand liberation of antitumor immune responses by transformation ofthe tumor microenvironment.

Disclosure of Potential Conflicts of InterestE. Quintana is Director at Revolution Medicines. C.J. Schulze is Senior Scientist at

Revolution Medicines. D.R. Myers is Scientist at Revolution Medicines. T.J. Choy isResearch Associate II at Revolution Medicines. K. Mordec is Senior ResearchAssociate Scientist at Revolution Medicines. D. Wildes is Senior Director/PrincipalScientist at Revolution Medicines. N. Tobvis Shifrin is Sr. Scientist at Revolution

Medicines. A. Belwafa is Senior Research Associate at Revolution Medicines.E.S. Koltun is Senior Director Medicinal Chemistry at and has an ownership interest(including patents) in Revolution Medicines. A.L. Gill is Senior Vice President,Chemistry, at RevolutionMedicines. M. Singh is Sr. Director at and has an ownershipinterest (including patents) in Revolution Medicines. S. Kelsey is President, Researchand Development, at and has an ownership interest (including patents) in RevolutionMedicines. M.A. Goldsmith is CEO at Revolution Medicines. R. Nichols is Director,Biology, at and has an ownership interest (including patents) in Revolution Med-icines. J.A.M. Smith is Sr. Vice President, Biology, at Revolution Medicines. Nopotential conflicts of interest were disclosed by the other authors.

Authors’ ContributionsConception and design: E. Quintana, C.J. Schulze, A.L. Gill, M. Singh, S. Kelsey,M.A. Goldsmith, J.A.M. SmithDevelopment of methodology: D.R. Myers, D. Wildes, A.L. Gill, J.A.M. SmithAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): C.J. Schulze, T.J. Choy, K. Mordec, D. Wildes, N. Tobvis Shifrin,A. Belwafa, E.S. Koltun, M. Singh, J.A.M. SmithAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): E. Quintana, C.J. Schulze, D.R. Myers, T.J. Choy,K. Mordec, D. Wildes, A. Belwafa, S. Kelsey, M.A. Goldsmith, J.A.M. SmithWriting, review, and/or revision of the manuscript: E. Quintana, C.J. Schulze,D.R. Myers, T.J. Choy, D. Wildes, N. Tobvis Shifrin, M. Singh, S. Kelsey,M.A. Goldsmith, R. Nichols, J.A.M. SmithStudy supervision: E. Quintana, J.A.M. SmithOther (key tool compound design and synthesis): E.S. Koltun

AcknowledgmentsWe would like to thank Dylan Daniel, Art Weiss, and Cliff Lowell for providing

expert advice during the course of this work. We would also like to thank therespective research teams at the following contract research organizations for theconduct of in vitro and in vivo studies: MI Bioresearch, HDB,WuXI Apptec, Ensigna,HistoTox Labs, and PAIRimmune, Inc. This work was supported in part by Sanofi.

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 October 1, 2019; revised February 23, 2020; accepted April 22, 2020;published first April 29, 2020.

References1. Nichols RJ, Haderk F, Stahlhut C, Schulze CJ, Hemmati G, Wildes D, et al. RAS

nucleotide cycling underlies the SHP2 phosphatase dependence of mutantBRAF-, NF1- and RAS-driven cancers. Nat Cell Biol 2018;20:1064–73.

2. Ruess DA, Heynen GJ, Ciecielski KJ, Ai J, Berninger A, Kabacaoglu D, et al.Mutant KRAS-driven cancers depend on PTPN11/SHP2 phosphatase. Nat Med2018;24:954–60.

3. Mainardi S,Mulero-SanchezA, PrahalladA,GermanoG, BosmaA,KrimpenfortP, et al. SHP2 is required for growth of KRAS-mutant non-small-cell lung cancerin vivo. Nat Med 2018;24:961–7.

4. ChenYN, LaMarcheMJ, ChanHM, Fekkes P,Garcia-Fortanet J, AckerMG, et al.Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptortyrosine kinases. Nature 2016;535:148–52.

5. Frankson R, Yu ZH, Bai Y, Li Q, Zhang RY, Zhang ZY. Therapeutic targeting ofoncogenic tyrosine phosphatases. Cancer Res 2017;77:5701–5.

6. Fedele C, RanH, Diskin B,WeiW, Jen J, GeerMJ, et al. SHP2 inhibition preventsadaptive resistance to MEK inhibitors in multiple cancer models. Cancer Discov2018;8:1237–49.

7. Ahmed TA, Adamopoulos C, Karoulia Z, Wu X, Sachidanandam R, AaronsonSA, et al. SHP2 drives adaptive resistance to erk signaling inhibition in molec-ularly defined subsets of ERK-dependent tumors. Cell Rep 2019;26:65–78.

8. Ou S-HI. A12 the SHP2 inhibitor RMC-4630 in patients with KRAS-mutantnon-small cell lung cancer: preliminary evaluation of a first-in-man phase 1clinical trial. J Thorac Oncol 2020;15:S15–S16.

9. Zhao M, Guo W, Wu Y, Yang C, Zhong L, Deng G, et al. SHP2 inhibitiontriggers anti-tumor immunity and synergizes with PD-1 blockade. ActaPharmaceuticaSinicaB 2019;9:304–15.

10. Liu W, Guo W, Shen L, Chen Z, Luo Q, Luo X, et al. T lymphocyte SHP2-deficiency triggers anti-tumor immunity to inhibit colitis-associated cancer inmice. Oncotarget 2017;8:7586–97.

11. Rota G, Niogret C, Dang AT, Barros CR, Fonta NP, Alfei F, et al. Shp-2 isdispensable for establishing T cell exhaustion and for PD-1 signaling in vivo.Cell Rep 2018;23:39–49.

12. Xiao P, Guo Y, Zhang H, Zhang X, Cheng H, Cao Q, et al. Myeloid-restrictedablation of Shp2 restrains melanoma growth by amplifying the reciprocalpromotion of CXCL9 and IFN-gamma production in tumormicroenvironment.Oncogene 2018;37:5088–100.

13. Pathria P, Louis TL, Varner JA. Targeting tumor-associated macrophages incancer. Trends Immunol 2019;40:310–27.

14. PedersenMB, Danielsen AV, Hamilton-Dutoit SJ, Bendix K, Norgaard P,MollerMB, et al. High intratumoral macrophage content is an adverse prognosticfeature in anaplastic large cell lymphoma. Histopathology 2014;65:490–500.

15. Jackute J, Zemaitis M, Pranys D, Sitkauskiene B, Miliauskas S, Vaitkiene S, et al.Distribution ofM1 andM2macrophages in tumor islets and stroma in relation toprognosis of non-small cell lung cancer. BMC Immunol 2018;19:3.

16. Shigeoka M, Urakawa N, Nakamura T, Nishio M, Watajima T, Kuroda D, et al.Tumor associated macrophage expressing CD204 is associated with tumoraggressiveness of esophageal squamous cell carcinoma. Cancer Sci 2013;104:1112–9.

17. Kim KJ, Wen XY, Yang HK, KimWH, Kang GH. Prognostic implication of M2macrophages are determined by the proportional balance of tumor associatedmacrophages and tumor infiltrating lymphocytes in microsatellite-unstablegastric carcinoma. PLoS One 2015;10:e0144192.

Quintana et al.





18. Bostrom MM, Irjala H, Mirtti T, Taimen P, Kauko T, Algars A, et al. Tumor-associated macrophages provide significant prognostic information in urothelialbladder cancer. PLoS One 2015;10:e0133552.

19. Guo B, Cen H, Tan X, Ke Q.Meta-analysis of the prognostic and clinical value oftumor-associatedmacrophages in adult classical Hodgkin lymphoma. BMCMed2016;14:159.

20. Yin S, Huang J, Li Z, Zhang J, Luo J, Lu C, et al. The prognostic andclinicopathological significance of tumor-associated macrophages in patientswith gastric cancer: a meta-analysis. PLoS One 2017;12:e0170042.

21. Morita Y, ZhangR, LeslieM,Adhikari S,HasanN,Chervoneva I, et al. Pathologicevaluation of tumor-associated macrophage density and vessel inflammation ininvasive breast carcinomas. Oncol Lett 2017;14:2111–8.

22. Zhang QW, Liu L, Gong CY, Shi HS, Zeng YH, Wang XZ, et al. Prognosticsignificance of tumor-associated macrophages in solid tumor: a meta-analysis ofthe literature. PLoS One 2012;7:e50946.

23. Cassetta L, Kitamura T. Targeting tumor-associated macrophages as a potentialstrategy to enhance the response to immune checkpoint inhibitors. Front CellDev Biol 2018;6:38.

24. Gavrieli M, Watanabe N, Loftin SK, Murphy TL, Murphy KM. Characterizationof phosphotyrosine binding motifs in the cytoplasmic domain of B and Tlymphocyte attenuator required for association with protein tyrosine phospha-tases SHP-1 and SHP-2. Biochem Biophys Res Commun 2003;312:1236–43.

25. Sheppard KA, Fitz LJ, Lee JM, Benander C, George JA, Wooters J, et al. PD-1inhibits T-cell receptor induced phosphorylation of the ZAP70/CD3zeta signa-losome and downstream signaling to PKCtheta. FEBS Lett 2004;574:37–41.

26. Marasco M, Berteotti A, Weyershaeuser J, Thorausch N, Sikorska J, Krausze J,et al. Molecular mechanism of SHP2 activation by PD-1 stimulation. Sci Adv2020;6:eaay4458.

27. Chemnitz JM, Parry RV, Nichols KE, June CH, Riley JL. SHP-1 and SHP-2associate with immunoreceptor tyrosine-based switch motif of programmeddeath 1 upon primary human T cell stimulation, but only receptor ligationprevents T cell activation. J Immunol 2004;173:945–54.

28. Hui E, Cheung J, Zhu J, Su X, Taylor MJ, Wallweber HA, et al. T cellcostimulatory receptor CD28 is a primary target for PD-1-mediated inhibition.Science 2017;355:1428–33.

29. Latchman Y, Wood CR, Chernova T, Chaudhary D, Borde M, Chernova I, et al.PD-L2 is a second ligand for PD-1 and inhibits T cell activation. Nat Immunol2001;2:261–8.

30. Peled M, Tocheva AS, Sandigursky S, Nayak S, Philips EA, Nichols KE, et al.Affinity purification mass spectrometry analysis of PD-1 uncovers SAP as a newcheckpoint inhibitor. Proc Natl Acad Sci U S A 2018;115:E468–e77.

31. Yamamoto R, Nishikori M, Kitawaki T, Sakai T, HishizawaM, TashimaM, et al.PD-1-PD-1 ligand interaction contributes to immunosuppressive microenvi-ronment of Hodgkin lymphoma. Blood 2008;111:3220–4.

32. Okazaki T,MaedaA,NishimuraH, Kurosaki T, Honjo T. PD-1 immunoreceptorinhibits B cell receptor-mediated signaling by recruiting src homology2-domain-containing tyrosine phosphatase 2 to phosphotyrosine. Proc NatlAcad Sci U S A 2001;98:13866–71.

33. Yokosuka T, TakamatsuM,Kobayashi-ImanishiW,Hashimoto-TaneA, AzumaM, Saito T. Programmed cell death 1 forms negative costimulatorymicroclustersthat directly inhibit T cell receptor signaling by recruiting phosphatase SHP2.J Exp Med 2012;209:1201–17.

34. Marengere LE, Waterhouse P, Duncan GS, Mittrucker HW, Feng GS, Mak TW.Regulation of T cell receptor signaling by tyrosine phosphatase SYP associationwith CTLA-4. Science 1996;272:1170–3.

35. Teft WA, Kirchhof MG, Madrenas J. A molecular perspective of CTLA-4function. Annu Rev Immunol 2006;24:65–97.

36. Wang B, Zhang W, Jankovic V, Golubov J, Poon P, Oswald EM, et al. Com-bination cancer immunotherapy targeting PD-1 andGITR can rescue CD8(þ) Tcell dysfunction and maintain memory phenotype. Sci Immunol 2018;3:eaat7061.

37. Celis-Gutierrez J, Blattmann P, Zhai Y, Jarmuzynski N, Ruminski K, Gregoire C,et al. Quantitative interactomics in primary T cells provides a rationale forconcomitant PD-1 and BTLA coinhibitor blockade in cancer immunotherapy.Cell Rep 2019;27:3315–30.

38. Hao HX, Wang H, Liu C, Kovats S, Velazquez R, Lu H, et al. Tumor intrinsicefficacy by SHP2 andRTK inhibitors in KRAS-mutant cancers.Mol Cancer Ther2019;18:2368–80.

39. Barber DL, Wherry EJ, Masopust D, Zhu B, Allison JP, Sharpe AH, et al.Restoring function in exhausted CD8 T cells during chronic viral infection.Nature 2006;439:682–7.

40. Kamphorst AO,WielandA, Nasti T, Yang S, Zhang R, Barber DL, et al. Rescue ofexhausted CD8 T cells by PD-1-targeted therapies is CD28-dependent. Science2017;355:1423–7.

41. DeNardo DG, Ruffell B. Macrophages as regulators of tumour immunity andimmunotherapy. Nat Rev Immunol 2019;19:369–82.

42. Gubin MM, Esaulova E, Ward JP, Malkova ON, Runci D, Wong P, et al. High-dimensional analysis delineates myeloid and lymphoid compartment remodel-ing during successful immune-checkpoint cancer therapy. Cell 2018;175:1014–30.e19.

43. Xiong H, Mittman S, Rodriguez R, Moskalenko M, Pacheco-Sanchez P, Yang Y,et al. Anti-PD-L1 treatment results in functional remodeling of the macrophagecompartment. Cancer Res 2019;79:1493–506.

44. Na YR, Jung D, Gu GJ, Seok SH. GM-CSF grown bone marrow derived cells arecomposed of phenotypically different dendritic cells andmacrophages.Mol Cells2016;39:734–41.

45. Pixley FJ, Stanley ER. CSF-1 regulation of the wandering macrophage: com-plexity in action. Trends Cell Biol 2004;14:628–38.

46. Cannarile MA, Weisser M, Jacob W, Jegg AM, Ries CH, Ruttinger D. Colony-stimulating factor 1 receptor (CSF1R) inhibitors in cancer therapy.J Immunother Cancer 2017;5:53.

47. Pyonteck SM, Akkari L, Schuhmacher AJ, Bowman RL, Sevenich L, Quail DF,et al. CSF-1R inhibition alters macrophage polarization and blocks gliomaprogression. Nat Med 2013;19:1264–72.

48. Neubert NJ, SchmittnaegelM, Bordry N, Nassiri S,WaldN,Martignier C, et al. Tcell-induced CSF1 promotes melanoma resistance to PD1 blockade. Sci TranslMed 2018;10.

49. Lee KM, Chuang E, Griffin M, Khattri R, Hong DK, Zhang W, et al. Molecularbasis of T cell inactivation by CTLA-4. Science 1998;282:2263–6.

50. Wang L, Iorio C,YanK, YangH, Takeshita S, Kang S, et al. A ERK/RSK-mediatednegative feedback loop regulatesM-CSF-evoked PI3K/AKT activation inmacro-phages. FASEB J 2018;32:875–87.

51. Zhu Y, Knolhoff BL, Meyer MA, Nywening TM, West BL, Luo J, et al. CSF1/CSF1R blockade reprograms tumor-infiltrating macrophages and improvesresponse to T-cell checkpoint immunotherapy in pancreatic cancer models.Cancer Res 2014;74:5057–69.

52. Ries CH, Cannarile MA, Hoves S, Benz J, Wartha K, Runza V, et al. Targetingtumor-associated macrophages with anti-CSF-1R antibody reveals a strategy forcancer therapy. Cancer Cell 2014;25:846–59.

53. Van Overmeire E, Stijlemans B, Heymann F, Keirsse J, Morias Y, Elkrim Y, et al.M-CSF and GM-CSF receptor signaling differentially regulate monocyte mat-uration and macrophage polarization in the tumor microenvironment.Cancer Res 2016;76:35–42.

54. Ao JY, Zhu XD, Chai ZT, Cai H, Zhang YY, Zhang KZ, et al. Colony-stimulatingfactor 1 receptor blockade inhibits tumor growth by altering the polarization oftumor-associated macrophages in hepatocellular carcinoma. Mol Cancer Ther2017;16:1544–54.

55. Tsai CC, Kai JI, Huang WC, Wang CY, Wang Y, Chen CL, et al. Glycogensynthase kinase-3beta facilitates IFN-gamma-induced STAT1 activation byregulating Src homology-2 domain-containing phosphatase 2. J Immunol2009;183:856–64.

56. Baron M, Davignon JL. Inhibition of IFN-gamma-induced STAT1 tyrosinephosphorylation by human CMV is mediated by SHP2. J Immunol 2008;181:5530–6.

57. Wu TR, Hong YK, Wang XD, Ling MY, Dragoi AM, Chung AS, et al.SHP-2 is a dual-specificity phosphatase involved in Stat1 dephosphory-lation at both tyrosine and serine residues in nuclei. J Biol Chem 2002;277:47572–80.

58. HuX, Ivashkiv LB. Cross-regulation of signaling pathways by interferon-gamma:implications for immune responses and autoimmune diseases. Immunity 2009;31:539–50.

59. Strauss L,MahmoudMAA,Weaver JD, Tijaro-OvalleNM,ChristofidesA,WangQ, et al. Targeted deletion of PD-1 inmyeloid cells induces antitumor immunity.Sci Immunol 2020;5:eaay1863.

60. Cassetta L, Fragkogianni S, Sims AH, Swierczak A, Forrester LM, Zhang H, et al.Human tumor-associatedmacrophage andmonocyte transcriptional landscapesreveal cancer-specific reprogramming, biomarkers, and therapeutic targets.Cancer Cell 2019;35:588–602.e10.

61. Christensen JG, Fell JB, Marx MA, Fischer J, Hallin J, Calinisan BB, et al.Insight towards therapeutic susceptibility of KRAS mutant cancers fromMRTX1257, a novel KRAS G12C mutant selective small molecule inhibitor[abstract]. In: Proceedings of the 110th Annual Meeting of the American






Association for Cancer Research; 2019 March 29–Apr 3; Atlanta GA; CancerRes 2019;79(13_Suppl): Abstract nr LB-271.

62. Dardaei L, Wang HQ, Singh M, Fordjour P, Shaw KX, Yoda S, et al. SHP2inhibition restores sensitivity in ALK-rearranged non-small-cell lung cancerresistant to ALK inhibitors. Nat Med 2018;24:512–7.

63. Lee GL, Stahlhut C, Evans J, Reyes DF, Lorenzana EG, L. S, et al. Maximizing thetherapeutic potential of SHP2 inhibition with rational combination strategies intumors driven by aberrant RAS-MAPK signaling [abstract]. In: Proceedings of the110th Annual Meeting of the American Association for Cancer Research; 2019March 29–Apr 3; Atlanta GA; Cancer Res 2019;79(13_Suppl): Abstract nr 1322.


Quintana et al.




2020;80:2889-2902. Published OnlineFirst April 29, 2020.Cancer Res Elsa Quintana, Christopher J. Schulze, Darienne R. Myers, et al. Transforming the Immunosuppressive EnvironmentAllosteric Inhibition of SHP2 Stimulates Antitumor Immunity by

Updated version

10.1158/0008-5472.CAN-19-3038doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2020/04/28/0008-5472.CAN-19-3038.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/80/13/2889.full#ref-list-1

This article cites 60 articles, 23 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/80/13/2889.full#related-urls

This article has been cited by 2 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/80/13/2889To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-19-3038

http://cancerres.aacrjournals.org/content/suppl/2020/04/28/0008-5472.CAN-19-3038.DC1

http://cancerres.aacrjournals.org/content/80/13/2889.full#ref-list-1

http://cancerres.aacrjournals.org/content/80/13/2889.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/80/13/2889


allosteric inhibition of shp2 stimulates antitumor ... · allosteric inhibition of the protein...

Documents