Allosteric inhibition of SHP2 stimulates anti-tumor …...2020/04/28 · Tumor Immunity by Transforming the Immunosuppressive Environment Elsa Quintana1, Christopher J. Schulze1,

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Allosteric inhibition of SHP2 Stimulates Anti-Tumor Immunity by

Transforming the Immunosuppressive Environment

Elsa Quintana1, Christopher J. Schulze

1, Darienne R. Myers

1, Tiffany J. Choy

1, Kasia Mordec

1,

David Wildes1, Nataliya Tobvis Shifrin

1, Amira Belwafa

1, Elena S. Koltun

2, Adrian L. Gill

2,

Mallika Singh1, Stephen Kelsey

1,2, Mark A. Goldsmith

1,2, Robert Nichols

1, Jacqueline A. M.

Smith1*

1 Department of Biology, Revolution Medicines, Inc., Redwood City, CA, USA.

2 Department of Chemistry, Revolution Medicines, Inc., Redwood City, CA, USA.

*e-mail: [email protected]

*Address: 700 Saginaw Drive, Redwood City, CA 94063

*Phone: 650-481-6920

Running Title

SHP2 in macrophages and tumor immunosuppression

The authors are full time employees of Revolution Medicines and declare no competing financial

interests.

on June 1, 2020. © 2020 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 29, 2020; DOI: 10.1158/0008-5472.CAN-19-3038

mailto:[email protected]

http://cancerres.aacrjournals.org/

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Abstract The protein-tyrosine phosphatase SHP2 binds to phosphorylated signaling motifs on regulatory

immunoreceptors including PD-1, but its functional role in tumor immunity is unclear. Using

preclinical models, we show that RMC-4550, an allosteric inhibitor of SHP2, induces anti-tumor

immunity with effects equivalent to or greater than those resulting from checkpoint blockade. In

the tumor microenvironment, inhibition of SHP2 modulated T cell infiltrates similar to

checkpoint blockade. Additionally, RMC-4550 drove direct, selective depletion of pro-

tumorigenic M2 macrophages via attenuation of CSF-1 receptor signaling and increased M1

macrophages via a mechanism independent of CD8+T-cells or IFN-γ. These dramatic shifts in

polarized macrophage populations in favor of anti-tumor immunity were not seen with

checkpoint blockade. Consistent with a pleiotropic mechanism of action, RMC-4550 in

combination with either checkpoint or CSF-1R blockade caused additive anti-tumor activity with

complete tumor regressions in some mice; tumors intrinsically sensitive to SHP2 inhibition or

checkpoint blockade were particularly susceptible. Our preclinical findings demonstrate that

SHP2 thus plays a multifaceted role in inducing immune suppression in the tumor

microenvironment, through both targeted inhibition of RAS pathway-dependent tumor growth

and liberation of anti-tumor immune responses. Furthermore, these data suggest that inhibition

of SHP2 is a promising investigational therapeutic approach.

Significance Inhibition of SHP2 causes direct and selective depletion of pro-tumorigenic M2 macrophages

and promotes anti-tumor immunity, highlighting an investigational therapeutic approach for

some RAS pathway-driven cancers.




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Introduction

Allosteric 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 therapeutic strategy for patients bearing tumors with specific oncogenic mutations

in this pathway (1-4). SHP2 is a positive transducer of receptor tyrosine kinase (RTK) signaling

(see Frankson et al. for a recent (5) review) but the molecular mechanism is still unclear. We and

others have shown that SHP2 acts upstream of RAS and promotes RTK-mediated RAS

nucleotide exchange and activation, likely through a scaffolding interaction with SOS1(1,6,7).

Clinical studies with investigational SHP2 inhibitors are ongoing and preliminary signs of

clinical activity in patients with non-small cell lung cancer (NSCLC) harboring KRAS

mutations, particularly KRASG12C

, have been reported (8). SHP2 is also widely expressed in

hematopoietic cells, including both lymphoid and myeloid cells, and there is emerging evidence

to support a role in anti-tumor immunity. The majority of reported 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 melanoma growth (12).

Tumor-associated myeloid cell infiltration is associated with clinical resistance to

immunotherapies (13) and correlates with a negative prognosis for most tumor types (14-22).

Identification of therapeutic strategies that can modulate the recruitment, survival and/or

reprograming of tumor-associated macrophages (TAMs) and improve the clinical response to

currently available immunotherapies is critical (23). Building a comprehensive understanding of

the impact, if any, 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 targeted therapeutic strategy.

SHP2 may also be an important signaling node downstream of inhibitory receptors in

immune cells. SHP2 binds to tandem phosphorylated ITIM (immunoreceptor tyrosine-based

inhibition motif) and ITSM (immunoreceptor tyrosine-based switch motif) domains on

regulatory receptors in immune cells, including inhibitory immune receptors like PD-1 and

BTLA (24-26), and multiple reports have demonstrated a SHP2/PD-1 physical interaction in

vitro (25,27-33). Regulation of T-cell receptor signaling in vitro by SHP2 association with

CTLA4 has also been reported (34) although canonical ITIM/ITSM domains are not present in

CTLA4, so the significance of these reported associations is unclear (35). More recently, through

the application of cell-free biochemical experiments, it has been proposed that SHP2 transduces

the PD-1 inhibitory checkpoint signal by direct de-phosphorylation of the co-stimulatory

molecules CD28 and CD226 and, consequently, limits T-cell activation (28,36). Collectively

these studies have pointed to a role for SHP2 in regulation of T-cells. However, using a T-cell-

specific SHP2 deficient mouse model, Rota et al. concluded that SHP2 is dispensable for PD-1

signaling in T-cells in vivo, as well as for the global induction of T-cell exhaustion (11), a

process that PD-1 has been implicated in controlling. Furthermore, the control of immunogenic

tumors was not improved in these T-cell-SHP2-deficient mice, and the response to anti-PD-1

checkpoint blockade therapy was not affected (11). One plausible explanation for the apparent

discrepancy between these observations is that redundant mechanisms, such as the related

tyrosine phosphatase SHP1, can mediate PD-1 inhibitory signaling in the setting of SHP2

deficiency (37). The emergence of these types of compensatory signaling mechanisms highlights

the limitations of using genetically-engineered mouse models to interrogate the in vivo

mechanism(s) of action of SHP2. Moreover, the selective deletion of SHP2 protein from only a

subset of immune cells obscures the clinical implications of the findings thus far, as it does not




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appropriately model the effects of pharmacological inhibition of SHP2 broadly 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 to interrogate the immunomodulatory mechanism(s)

of action of SHP2 in vivo using pharmacological tools that circumvent the various limitations

imposed by genetic approaches. Accordingly, Zhao et al. have reported that a selective, but low

potency, small molecule inhibitor of SHP2 decreases tumor burden by augmenting cytotoxic T-

cell-mediated anti-tumor immunity (9). However, in this study no evidence was provided to

substantiate a direct effect of the SHP2 inhibitor on T-cells in vivo, and further the impact of

SHP2 inhibition on the myeloid compartment was not evaluated (9).

In this study, we used the previously described potent and selective allosteric inhibitor of

SHP2, RMC-4550 (1), to generate an in-depth understanding of the integrated effects of SHP2

inhibition in vivo in the tumor microenvironment. Using syngeneic mouse models we reveal an

unanticipated impact of SHP2 on tumor immunity through modulation of both innate and

adaptive immune cells. Similar to immune checkpoint blockade, RMC-4550 caused an increase

in CD8+ T-cell tumor infiltrates. RMC-4550 also produced a direct and selective depletion of

pro-tumorigenic M2 macrophages through attenuation of CSF-1 receptor (CSF-1R) signaling.

The anti-tumor effects of RMC-4550 were additive with either immune checkpoint inhibitors or

an anti-CSF-1R antibody, consistent with a pleiotropic role for SHP2 in the tumor

microenvironment. Tumors that are intrinsically sensitive to SHP2 inhibition and also sensitive

to checkpoint blockade were particularly susceptible to RMC-4550 alone or the combination

treatment.

Collectively these findings highlight that SHP2 inhibition is a promising molecular

therapeutic strategy in cancer with potential dual activity: targeted suppression of tumor-intrinsic

RAS/MAPK dependent growth and promotion of anti-tumor immune responses through

transformation of the suppressive tumor immune microenvironment. Translation of the

preclinical combination advantages of a SHP2 inhibitor and checkpoint blockade into the clinical

setting would be a significant advance for patients bearing oncogenic RAS pathway alterations

and for whom current therapeutic options and benefits are limited.

Materials and Methods

Cell Lines and Reagents. All cell lines were obtained from ATCC except for MC38

(NTCCChina). Cells were grown in RPMI (CT26.WT, A20 and 4T1) or DMEM (MC38, EMT6,

B16-F10) supplemented with 10% heat inactivated fetal bovine serum and 1%

penicillin/streptomycin (Gibco). Cells were maintained at 37 °C in a humidified incubator at 5%

CO2. All cells were mycoplasma free and identity was confirmed by short tandem repeat

profile. Antibodies used for in vivo treatment were from BioXcell: anti-PD-L1 (10F.9G2), rat

IgG2b (LFT-2); anti-PD1 (RMP1-14), rat IgG2a (2A3), anti-CTLA4 (9D9), mIgG2b (MPC11);

anti-CSF-1R (AFS98), rat IgG2a (2A3), anti-CD4 (GK1.5) and anti-CD8 (2.43).

In vivo tumor challenge. All studies were compliant with all relevant ethical regulations

regarding animal research in accordance with approved institutional animal care and use

committee IACUC protocols at MI Bioresearch, Inc. (Ann Arbor, MI), WuXi Apptec. (Suzhou,

China) and HD Biosciences (San Diego, CA). Female (6-8 weeks old) immunocompetent mice




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were implanted subcutaneously with 5E+05 CT26.WT cells, 5E+05 A20 cells or 5E+05 EMT6

cells (BALB/c, Envigo or Balb/c Rag2 ko/ko, Taconic), and 2E+05 MC38 or 5E+04 B16-F10

cells (C57BL/6J, SLAC Laboratory Animal Co., LTD.); or injected in the mammary fat pad with

5E+05 4T1 cells (BALB/c, Shanghai Lingchang Biological Technology Co., LTD.). Once

tumors reached an average size of 48 to 90 mm3, administration of RMC-4550 (30 mg kg−1, by

daily oral administration) or vehicle (2% HPMC in 50mM sodium citrate buffer), anti-PD-1,

anti-PD-L1 or anti-CTLA4 (10 mg kg−1, by intraperitoneal administration every three days), or

anti-CSF-1R (2 mg per mouse on day 1 followed by 0.2 mg per mouse 16 days after cell

implantation, by intraperitoneal administration), was initiated. Experiments with A20 or CT26

where anti-PD-L1 was investigated were not staged and treatment started 3 days after cell

implantation. The selective depletion of tissue macrophages by anti-CSF1R administration was

confirmed in liver by flow cytometry.

In vivo immune cell depletion experiments. Treatment began on day 7 at an overall mean

tumor burden of 72 mm3. Anti-CD4, anti-CD8 or combination were administered

intraperitoneally (0.5 mg per mouse on days 7, 8 and 9 followed by 0.2 mg per mouse on days 13

and 17). RMC-4550 was administered orally (30 mg kg−1 daily during 21 days starting at day

9). Depletion of immune cells in blood was confirmed by flow cytometry.

Immune phenotyping studies in tumors. Treatment with anti-PD-L1 (10 mg kg−1

intraperitoneal on days 3, 6, 10 and 13), RMC-4550 (30 mg kg−1, by oral daily on days 3 to 15)

or combination started on day 3 and tumors were processed for analysis on day 16 after cell

implantation. Treatment with anti-CTLA4 (10 mg kg−1, intraperitoneal on days 7, 10 and 14),

RMC-4550 (30 mg kg−1, daily oral on days 7 to 15) or the combination started on day 7 (79

mm3 tumors) and tumors were processed for analysis on day 16. Tumors were dissociated into

single cell suspension (GentleMACS 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, Bbiolegend), MHC

Class II (proprietary from MI Bioresearch), CD45 (30-F11, BD Biosciences), CD206 (CO68C2,

Biolegend), CD11c (N418, ThermoFisher), Ly6G (1A8, BD Biosciences), CD19 (1D3, BD

Biosciences) and PD-L1 (B7H1, Biolegend), Ki67 (Biolegend, 16A8), AH1 Dextramer

(Immudex JG3294). ACK Lysing Buffer (Biolegend), Zombie Viability Dye (Biolegend), Fc

blocking agent (anti-CD16/32, Biolegend), FoxP3 Fix/Perm kit (eBiosciences), AbC Total

compensation (ThermoFisher), Cell staining buffer (BD Biosciences) were used. Samples were

run in an Attune NxT flow cytometer.

Immunohistochemistry detection for CD8a, F4/80, in mouse paraffin embedded tumors.

Anti-mouse CD8a (Cell Signaling, 98941, 1.6 µg/ml) or anti-F4/80 (Cell Signaling, 70076, 1.4

µg/ml) rabbit monoclonal antibodies were used with citrate-based pH 6.2 Heat-Induced Epitope

Retrieval. Sections (5 µm) were stained on the Biocare intelliPATH platform using the

manufacturer’s recommended settings. Antibody binding was detected with MACH4 HRP-

polymer Detection System followed by IntelliPATH FLX DAB chromogen and IntelliPATH

Hematoxylin kits. All reagents were from Biocare Medical, Pacheco CA. TissueScope LE whole




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slide scanner (Huron Digital Pathology), Huron Viewer software and HALO® Image Analysis

software from Indica labs were used for analysis.

PD-1 NFAT Luciferase Reporter Assay. Engineered CHO-K1 cells (BPS Bioscience, 60536)

were incubated overnight in RPMI medium supplemented with 10% heat inactivated fetal bovine

serum and 1% penicillin/streptomycin (Gibco). Engineered Jurkat cells (BPS Bioscience, 60535)

were pre-incubated with RMC-4550 or anti-PD-1 for 30 minutes and added to CHO-K1 cells.

After 16 h, One-Step Luciferase Assay system (BPS Bioscience, 60690) was added according to

manufacturer’s instructions, and luminescence was measured on an EnVision Multilabel Plate

Reader (Perkin Elmer).

Staphylococcus aureus Enterotoxin B Superantigen (SEB) T cell stimulation assay. Human

buffy coat was obtained from San Diego Blood Bank. PBMC were isolated using SpMateTM

(Stemcells) and treated with anti-PD-1 (S228P, Invivogen), isotype control (Human IgG4,

Invivogen), RMC-4550 or vehicle (0.2% DMSO) at concentrations indicated. 30 min to 1h after

compound treatment, cells were stimulated with SEB (0.1 g ml−1, Toxin Technologies),

followed by incubation in presence of SEB and compounds at 37°C 5% CO2 incubator for 3

days. IL-2 content was analyzed in supernatant by standard ELISA (Abcam) with Perkin Elmer

Envision Microplate Reader.

Mixed Lymphocyte Reaction (MLR). Monocytes were isolated from fresh PBMC from healthy

donors (EasySep monocyte enrichment kit, Stemcell). Monocytes were differentiated (3 days)

and matured (3 days) into monocyte-derived dendritic cells (Mo-DCs) by using Milteny Biotec

reagents. Cells were immunophenotyped with CD14, CD209 and CD83 and purity was

confirmed to be >90%. Responder CD3+ T cells were prepared from a different donor using a

negative selection kit (Stemcell) to obtain untouched T cells. Cells were co-cultured at a final

ratio of T cells to Mo-DCs of 10:1. Anti-PD-1 (S228P, Invivogen), isotype control (Human

IgG4, Invivogen), RMC-4550 or vehicle were incubated for 5 days and supernatants were

assessed for IFN- by ELISA (Invitrogen).

In vitro studies with murine bone marrow derived macrophages (BMDM). Culture of

BMDMs: Bone marrow (BM) was isolated from the femurs of Balb/C mice. BM cells were

plated in complete Alpha-MEM media (Gibco) containing 10% heat-inactivated FBS (VWR)

and 1% Pen-Strep (Corning) and supplemented with CSF-1 (Peprotech) at 10 ng/mL or GM-CSF

(Peprotech) at 25 ng/mL. Growth inhibition and apoptosis assays: After 7 days of culture, BM

cells were dissociated (Gibco Cell Dissociation Buffer) and plated with media containing CSF-1

at 10 ng/mL or GM-CSF at 25 ng/mL. After 3 hours, cells were treated with either RMC-4550 or

BLZ-945 (Selleckchem). Cell proliferation was measured 72 h after compound treatment using

the CellTiterGlo reagent (Promega). Caspase activity was measured using the caspase 3/7 Glow

kit (Promega) 48 h after compound treatment. Polarization assay: BM cells were isolated as

above and cultured in 10 ng/mL CSF-1 for 6 days. The appropriate cytokine (R&D Systems) was

added (M1: IFN-gamma: 20 ng/mL, LPS: 100 ng/mL M2: IL-4: 20 ng/mL) and cells were

cultured for an additional 24 hours. Polarized BMDMs were treated with compound in the

presence of CSF-1 and the appropriate cytokine and analyzed for cell proliferation and caspase

3/7 activity as described. pERK assay: BM cells were isolated as above and cultured in either

CSF-1 or GM-CSF for 7 days. Growth factor was removed overnight and cells were treated with




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compound for 1 hour. Cells were acutely stimulated with CSF-1 at 100 ng/mL or GM-CSF at 50

ng/mL for 10 minutes. pERK was analyzed using the AlphaLisa SureFire p-ERK1/2

Thr202/Tyr204 kit (PerkinElmer) according to the manufacturer’s instructions. pAkt was

assessed using the pAkt (Ser473) MSD kit (Meso Scale Diagnostics) according to the

manufacturer’s instructions.

Statistics. Quantitative data are presented as the mean ± standard deviation (s.d.) or the standard

error of the mean (s.e.m.), as specified in the figure legends. Statistical tests were performed

using GraphPad Prism 7.0. Two-sided Student’s t-tests were used for comparisons of the means

of data between two groups and one-way ANOVA with post-hoc Tukey’s test was used for

comparisons among multiple independent groups, unless otherwise specified. For animal studies,

animals were randomized before treatments, and all animals treated were included for the

analyses. P Value < 0,05 was considered significant.

Results

SHP2 inhibition induces anti-tumor immunity in vivo in checkpoint blockade-sensitive

tumors

We first examined the anti-tumor efficacy of the SHP2 inhibitor RMC-4550 in three syngeneic

tumor models that are partially sensitive to checkpoint blockade: A20 B-cell lymphoma and both

MC38 and CT26 colon carcinomas. RMC-4550 had a modest effect on growth of A20 cells in

3D in vitro culture but did not reduce the viability of MC38 or CT26 cancer cells at

concentrations achievable in vivo (1) (IC50 = 2 M, >10 M and 10 M respectively, Fig. S1A).

RMC-4550 did inhibit RAS-MAPK signaling, as measured by phosphorylated ERK (pERK)

levels, in A20 and MC38 cells (IC50 of 4 M and 22 nM, respectively, Fig. S1A) but not in CT26

cells (IC50 >10 M, Fig. S1A). Repeated oral daily dosing of RMC-4550 at 30 mg/kg

significantly slowed tumor growth in each of these models (Fig. 1A). No effect of RMC-4550 on

CT26 tumor growth was observed in vivo when tumors were established in RAG-2 deficient

mice, which lack T and B lymphocytes and are thus immunocompromised (Fig. 1B). These data,

together with the lack of in vitro effect on both viability and RAS-MAPK signaling in CT26 cells

(Fig. S1A), provided confidence that the observed efficacy in vivo was a function of SHP2-

mediated effects on immune cells in the tumor microenvironment. To corroborate these findings

we demonstrated that RMC-4550 did not inhibit tumor growth in immunocompetent mice when

both CD4+ and CD8+T-cells had been functionally depleted in vivo with blocking antibodies

(Fig. 1C). CD8+T-cell depletion alone completely abrogated the efficacy of RMC-4550,

indicating that these immune effector cells are essential for SHP2 inhibitor mediated anti-tumor

immunity (Fig. 1C). Depletion of CD4+T-cells inhibited tumor growth in vehicle-treated mice

and no further inhibition of growth was apparent with RMC-4550 (Fig. S1B).

SHP2 inhibition is additive in combination with a checkpoint inhibitor

RMC-4550 induced significant tumor growth inhibition of CT26 tumors that was superior to

anti-PD-L1 (Fig. 1D) and comparable to anti-CTLA4 (Fig. 1E). The combination of RMC-4550

with anti-PD-L1 demonstrated robust anti-tumor benefit as evidenced by a significant increase in

the time to reach endpoint tumor burden and by tumor regressions in 4 of 10 mice (Fig. 1D). In

contrast, RMC-4550 or anti-PD-L1 treatment alone did not result in any tumor-free animals.




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Tumor-free survivors (TFS) remained tumor-free for at least 40 days and, importantly, were

resistant to tumor re-implantation, suggesting long-lasting adaptive immunity (Fig. S1C). All

treatments, including the combination, were well tolerated (Fig. S1D). Similar effects were

observed with anti-CTLA4, although the combination regimen was less well tolerated (Fig. 1E,

Fig. S1E).

SHP2 inhibition does not confer sensitivity to checkpoint blockade in PD-1 refractory

models

We tested the hypothesis that combination treatment with a SHP2 inhibitor could confer

sensitivity to checkpoint blockade using two syngeneic models that are refractory to anti-PD-1

treatment, 4T1 and B16F10. 4T1 breast carcinoma is a RAS/MAPK-dependent syngeneic line

sensitive to both MEK (trametinib, IC50 3D-growth = 5 nM) and SHP2 (RMC-4550, IC50 3D-

growth = 33 nM, Fig S1A) inhibition in vitro, while B16-F10 melanoma cells are insensitive to

SHP2 inhibition in vitro (Fig. S1A). RMC-4550 did not increase sensitivity to anti-PD-1 in vivo

in either of these models, irrespective of whether the cells were sensitive (4T1, Fig. S1F) or

insensitive (B16-F10, Fig. S1G) to the tumor intrinsic effects of SHP2 inhibition on RAS/MAPK

signaling.

SHP2 inhibition stimulates adaptive immunity

Analysis of the immune landscape of CT26 tumors demonstrated that RMC-4550 treatment

increased the percentage of CD3+T-cells by two-fold, from a baseline of 8±3% of CD45+ tumor-

infiltrating leukocytes (TILs) (Fig. S2A and B). CD8+T-cell frequency was increased in tumors

from RMC-4550-treated mice, while there was no change in CD4+T or T-regulatory (Treg) cell

frequency (Fig. 2A and B). Furthermore, the CD8+T-cells expressed less of the inhibitory

molecule PD-1 (Fig. 2B). The increase in CD8+T cell frequency was comparable to that

observed with checkpoint blockade, and the combination of RMC-4550 with either anti-PD-L1

or anti-CTLA4 exhibited additivity (Fig. 2A and B). The combinatorial effect with anti-CTLA4

on CD8+T cells frequency was statistically significant (Fig. 2B). RMC-4550 and anti-CTLA4

treatment-evoked increases in CD8+ T-cells were mostly localized to the border of the tumor and

while an increase relative to vehicle control was apparent for each of the single agent treatment

regimens only in the case of the combination of RMC-4550 with checkpoint blockade did the

increase reach statistical significance (Fig. 2C).

Following RMC-4550 treatment a higher frequency of the CD8+ tumor-infiltrating T

cells were specific for the tumor-associated antigen AH1 (analyzed by dextramer staining) (Fig.

S2C) and exhibited an activated profile, as they were proliferative (Ki67 staining) and expressed

the cytotoxic cytokine IFN (intracellular staining) (Fig. S2C). These changes did not reach

statistical significance but collectively are consistent with functional activation of the CD8+

tumor-infiltrating T cells.

RMC-4550 also increased the expression of class I MHC molecules and PD-L1 in CD45-

negative tumor cells, similarly to anti-CTLA4 (Fig. 2D). These effects were dependent on IFN

and CD8+T cells, as they were abrogated by depletion with the corresponding antibodies in vivo

(Fig. 2D). Consistent with the lack of intrinsic effects of RMC-4550 on proliferation of CT26

cells in vitro, the proliferation of CT26 cells in vivo was not affected, as measured by ki67

staining (Fig S2D). Finally, consistent with previous report (38), SHP2 inhibition did not affect

CT26 tumor vascularization as analyzed by CD31 tissue staining (Fig. S2E).




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

The direct effects, if any, of SHP2 inhibition on T-cells were explored in vitro and in vivo and

were compared to those of immune checkpoint blockade. Focusing on the proposed role of SHP2

as a downstream transducer of PD-L1/PD-1 signaling (25,28) we obtained robust biochemical

evidence that the tandem phosphorylated ITIM and ITSM in PD-1 can activate the auto-inhibited

form of SHP2. Titration of purified full length SHP2 with a synthetic peptide that mimics the

PD-1 tandem phosphorylated ITIM/ITSM increased enzyme activity by 270-fold (EC50 of 3.2

nM) (Fig. S2F). The PD-1 peptide (10 nM) induced activation of the auto-inhibited form of

SHP2 was blocked by RMC-4550 with an IC50 of 7.1 nM. To monitor PD-L1/PD-1 signal

transduction in a cellular context we used a bioluminescent reporter assay in Jurkat T-cells.

These Jurkat cells were engineered to express human PD-1 and a luciferase reporter driven by an

NFAT response element, and were cocultured with a variant of CHO cells that can serve as

antigen-presenting cells (APCs). These APCs are CHO-K1 cells expressing human PD-L1 and

an engineered cell surface protein designed to activate cognate TCRs in an antigen-independent

manner. RMC-4550 caused a concentration-dependent activation of the NFAT luciferase

reporter with an apparent potency (EC50 = 3.5 nM) consistent with an on-mechanism effect for

the SHP2 inhibitor. However, the maximal signal induction was approximately four-fold lower

than that observed with anti-PD-1 (Fig. 2E). In human PBMC cultures both RMC-4550 and anti-

PD-1 enhanced IL-2 secretion in response to the superantigen SEB (staphylococcal enterotoxin

B), but the response to RMC-4550 was not prominent compared to that of anti-PD-1 (Fig. 2F

top). Furthermore, RMC-4550 (up to a test concentration of 5 M) did not elicit a response in

human T-cells during a mixed lymphocyte reaction, while anti-PD-1 produced a robust increase

in IFN release (Fig. 2F bottom).

Given the equivocal findings in vitro we elected to use an in vivo model to investigate the

role of SHP2 in PD-1 signaling. Checkpoint blockade has been shown to reduce CD8+ T-cell

exhaustion in different systems including the lymphocytic choriomeningitis virus (LCMV)

infection mouse model (39). A role for CD28-costimulation in CD8+T cell rescue in the LCMV

model has been confirmed (40) and we used this model to determine whether SHP2 inhibition

mimics the effects of anti-PD-1 on T-cell exhaustion and viral load reductions in vivo. In our

study, mice were challenged with LCMV clone 13 to establish a chronic infection, followed by

administration of RMC-4550 or anti-PD-L1. RMC-4550 induced a significant increase in the

frequency of CD8+T cells in the spleen, but this was not accompanied by a significant increase

in antigen-specific CD8+T cells. Ultimately, RMC-4550 failed to decrease viral titers in

peripheral organs (Fig. S2G). In contrast, anti-PD-L1 treatment did effectively increase antigenic

CD8+T cells, correlating with higher viral control in various organs (Fig. S2G).

In summary, while we cannot rule out a role for SHP2 downstream of PD-1 signaling we

have demonstrated, using various model systems in vitro and in vivo, that SHP2 inhibition and

PD-1 blockade are not equivalent with respect to direct modulation of T-cell function. Rather, it

seems likely that SHP2 inhibition can restrain PD-1 signaling to some extent, but that the full

downstream effects are blunted, potentially due to the recruitment of redundant signaling effector

molecules.

SHP2 inhibition modulates innate immunity, an effect not seen with checkpoint blockade

To explore additional mechanisms of SHP2 inhibitor action in vivo beyond transduction of

checkpoint signals, we focused on myeloid cells in the tumor microenvironment. CT26 tumors




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are rich in myeloid cells; CD11b+ cells constitute 79±2% of CD45+ TILs and 64±2% of those

are F4/80+ tumor associated macrophages (TAMs). RMC-4550 treatment had a striking impact

on tumor myeloid infiltrates, in particular macrophages, inducing a 3-fold decrease in the

frequency of F4/80+ cells amongst CD45+ TILs (Fig. 3A-C and Fig. S3A). This finding was

confirmed by immunohistochemical staining (Fig. 3D); the decrease in macrophages was most

evident in the core of the tumor (Fig.3D, Fig. S3B).

TAMs are highly plastic and can acquire different phenotypes in the tumor

microenvironment ranging from pro-inflammatory M1 TAMs (MHCIIhigh

and CD206 negative/low

)

to pro-tumorigenic M2 TAMs (MHCIIlow

and CD206high

) (41). RMC-4550 induced a significant

decrease in the frequency of M2, the predominant population in CT26 tumors (>90% of TAMs,

Fig. 3A-C), and an increase in the frequency of M1 amongst CD45+ TILs; by extension the

M2/M1 ratio was dramatically reduced (Fig. 3B-C). Checkpoint blockade elicited only a modest

effect on TAM frequencies (Fig. 3B-C) but the combination of checkpoint blockade and RMC-

4550 drove an even deeper modulation of TAMs (Fig. 3B-C). Checkpoint blockade previously

has been shown to modulate TAM frequencies indirectly, via modulation of CD8+T-cell

frequency and IFNγ secretion in the tumor microenvironment (42,43). In contrast, RMC-4550-

mediated modulation of M2-TAM frequencies was unchanged by depletion of effector cells or

IFNγ cytokine (Fig. 3E). As expected, IFNγ or CD8+T-cell depletion decreased the overall

frequency of M1-TAMs; however a significant, RMC-4550-mediated increase was still apparent

(Fig. 3E). The expression of MHCI in M1 and M2-TAM was significantly increased with RMC-

4550 treatment and this effect was dependent of IFN and CD8+T cells (Fig. 3F). The expression

of PD-L1 in tumor associated macrophages was not changed by RMC-4550 treatment (Fig.

S3E).

Granulocytic myeloid-derived suppressor cells (MDSC) (gMDSC) and monocytic MDSC

(mMDSC) accounted for 7±1% and 14±1% of CD45+ TILs, respectively. Treatment with RMC-

4550 increased the frequency of mMDSCs but had no effect on gMDSCs (Fig. 3G and H). The

expression of MHCI or PD-L1 in MDSC was not changed upon RMC-4550 treatment (Fig.

S3E). To explore potential functional consequences of a SHP2 inhibitor-mediated increase in

mMDSC we used an in vitro suppression assay. Co-culture of human MDSC with T-cells

induced suppression of T-cell proliferation and IFN release (Fig. S3F). RMC-4550 alone had no

effect on T-cell proliferation or cytokine release (Fig, S3F) but was able to block the anti-

proliferative effects of MDSCs on CD8+ T-cells (Fig. S3F). A concomitant concentration-

dependent increase in IFN release was also observed (Fig. S3F). The viability of MDSCs in

vitro was not affected by RMC-4550 (92,5-93.5% viable compared to 93.3% viable in DMSO-

treated MDSCs, determined by Flow cytometry).

The frequency of myeloid cells in spleen or peripheral blood of tumor bearing mice was

unchanged with RMC-4550 treatment, suggesting that myelopoiesis was not affected at this

timepoint (Fig. S3C,D).

In summary, SHP2 inhibition produces a marked shift in polarized macrophage populations in

the tumor microenvironment in favor of antitumor immunity an effect that was not observed

upon checkpoint blockade. This selective effect of RMC-4550 on myeloid cells may underlie the

combination benefit of a SHP2 inhibitor and checkpoint blockade on tumor growth inhibition

(Fig. 1D, E).




11

SHP2 inhibition suppresses CSF-1R signaling and selectively affects viability of M2

macrophages

The prominent reduction in macrophage frequency observed in vivo following administration of

RMC-4550, and the apparent lack of dependence on effector lymphocytes or cytokines, is

consistent with a direct effect of SHP2 inhibition on macrophage viability. To evaluate this

possibility, bone marrow (BM) cells from BALB/c mice were differentiated with colony-

stimulating factor 1 (CSF-1) or granulocyte-macrophage colony stimulating factor (GM-CSF) in

vitro. CSF-1 differentiated bone marrow-derived macrophages (BMDMs) represent a population

of F4/80+, MHCIIlow

, CD11c- macrophages, while GM-CSF differentiated BM cells are

MHCIIhigh

, CD11c+, and likely represent a mixture of macrophages and dendritic cells (44).

RMC-4550 potently inhibited the growth of CSF-1-differentiated (IC50 = 13 nM) but not GM-

CSF-differentiated (IC50 > 1 M) BM cells (Fig. 4A). Additionally, SHP2 inhibition selectively

induced caspase3/7 activation, as a marker of apoptosis, in CSF-1 differentiated BMDMs (EC50

= 2.8 nM, Fig. 4B).

The colony stimulating factor 1 receptor (CSF-1R) is a receptor tyrosine kinase (RTK)

that controls the survival and proliferation of macrophages (45) and is the target of several

therapeutic agents in clinical development for cancer (46). The selective CSF-1R kinase inhibitor

BLZ945 (47) also showed selective growth inhibition and induction of apoptosis in CSF-1-

differentiated, but not GM-CSF-differentiated, BMDMs (Fig. S4A,B). The time course of growth

inhibition by BLZ945 or RMC-4550 was similar, and comparable to that caused by CSF-1

deprivation (Fig. S4C). Given these observations, together with the well-established role of

SHP2 as a positive signal transducer downstream of many RTKs, we hypothesized that SHP2

inhibition suppresses CSF-1R signaling. Indeed, we observed strong inhibition of ERK 1/2

phosphorylation by RMC-4550 after acute stimulation of BMDMs with CSF-1 (IC50 = 3 nM,

Fig. 4C). These results were recapitulated using a recombinant cell line that reports on CSF-1R

activation and signaling (Fig. S4D). SHP2 inhibition also decreased GM-CSF induced ERK 1/2

phosphorylation, albeit to a lesser extent (IC50 = 93 nM, Fig. 4C), an effect which was not

observed with BLZ-945 (Fig. S4E) and likely accounts for the moderate growth inhibitory effect

of RMC-4550 in these cells. Importantly, these in vitro results were recapitulated in monocytes

purified from human PBMCs, with SHP2 inhibition resulting in decreased ERK 1/2

phosphorylation and potent inhibition of growth (IC50 = 35 nM) (Fig. 4D and S4F). Moderate

suppression of AKT phosphorylation, another important signaling node for survival downstream

of CSF-1R, was observed with RMC-4550 in human monocytes but not in murine BMDMs (Fig.

S4G-I).

BMDMs were polarized to either an M1 (IFN-, LPS) or M2 (IL-4) phenotype to explore

the contribution of a selective intrinsic effect of SHP2 inhibition on M2 macrophages over M1 in

vitro. The M1 polarized macrophages expressed higher levels of iNOS, whereas M2 polarization

resulted in increased levels of CD206 and arginase (Fig. S4J). M2 macrophage viability was

sensitive to RMC-4550 (IC50 = 19 nM) but M1 polarized macrophages remained almost entirely

refractory to drug treatment (IC50 > 1 M) (Fig. 4E). Similarly, SHP2 inhibition selectively

induced caspase 3/7 activation in M2 but not M1 macrophages (Fig. 4F), which likely accounts

for the dramatic decrease in M2 frequency observed in vivo (Fig. 3B).

We were unable to determine the impact of SHP2 inhibition on macrophage

differentiation per se because RMC-4550 produced a significant decrease in monocyte viability

when present during the differentiation, precluding robust phenotypic characterization of the

differentiated cells.




12

Recent data have suggested that increased levels of IFN- and TNF- in the tumor

microenvironment, caused by infiltration of CD8+ T-cells, can trigger an adaptive response of

CSF-1 production by certain cancer cells (48). This in turn can promote recruitment and

proliferation of immunosuppressive TAMs, hampering the anti-tumor immune response to

checkpoint inhibitors. Treatment of CT26 cells in vitro with IFN- and TNF- did increase

production of CSF-1 mRNA (Fig. S5A). However, we propose that the ability of RMC-4550 to

inhibit CSF-1R signaling and decrease immunosuppressive TAM populations, as shown herein,

would negate any inhibitory effects of CSF-1 release by tumor cells.

SHP2 inhibition exhibits greater anti-tumor activity relative to CSF-1R inhibition in vivo

The contribution of SHP2-mediated blockade of the CSF-1R signaling pathway to the anti-tumor

efficacy of RMC-4550 in the CT26 model was examined by comparing the response to that of

CSF-1R blockade. Anti-CSF-1R treatment, in contrast to RMC-4550, did not induce any

significant tumor growth delay (Fig. 4G-H). These findings provide evidence that the in vivo

anti-tumor immunomodulatory effects of a SHP2 inhibitor reflect more than modulation of the

myeloid compartment alone.

The combination of anti-CSF-1R and RMC-4550 showed additive anti-tumor effects in the CT26

model (Fig. 4G-H). While unexpected, this result may reflect the differential mechanisms of

inhibition of CSF-1R signaling by these two agents. Activation of parallel signaling pathways

downstream of CSF-1R (e.g. PI3K/Akt) is insensitive to SHP2 blockade (Fig. S4I), while direct

receptor inhibition likely suppresses additional pro-survival signaling pathways. Given the role

of both SHP2 and CSF-1R as key signaling nodes in multiple cell types and the complexity of

the tumor microenvironment in vivo, further studies are required to elucidate the precise

mechanism(s) underlying this combinatorial effect.

SHP2 inhibition is additive in combination with checkpoint blockade in a SHP2 inhibitor-

sensitive syngeneic model

The combined tumor-intrinsic and immune-mediated anti-tumor effects of SHP2

inhibition have not been reported. EMT6 breast carcinoma is a RAS/MAPK dependent

syngeneic line sensitive to both MEK (trametinib, IC50 3D-growth = 47 nM) and SHP2 (RMC-

4550, IC50 3D-growth = 100 nM, Fig. S1A) inhibition in vitro. RMC-4550 alone significantly

inhibited growth of established EMT6 tumors in immunocompetent mice in vivo, an effect

superior to that of anti-PD-1 (Fig. 5A). The combination of RMC-4550 and anti-PD-1 resulted in

sustained tumor growth inhibition that greatly increased the time to reach endpoint (Fig. 5B).

This treatment also led to tumor regressions in 20% of mice, which were resistant to tumor re-

implantation, suggestive of long-lasting adaptive immunity (Fig. S5B). Treatment of EMT6

tumor bearing mice with RMC-4550 also induced a significant reduction in tumor cell

proliferation, as measured by Ki67 staining, analyzed 9 days after treatment (Fig. S5C). These

data corroborate the findings of cell intrinsic effects of RMC-4550 on proliferation of EMT6

cells in vitro (Fig. S1A).

Based on the collective observations presented here, we propose a model in which the

pleiotropic effects of SHP2 inhibition on both innate and adaptive immunity cooperate to

enhance tumor cell elimination (Fig. 5C). This study reveals a direct role for SHP2 in supporting




13

an immunosuppressive tumor microenvironment in addition to an impact on pro-inflammatory

macrophages, although the mechanism underlying the effect on M1 macrophages is unclear. We

have demonstrated that CD8+T cells are obligatory for the anti-tumor activity of SHP2

inhibition; however, the underlying mechanistic driver(s) of the augmented adaptive immune

response remains to be determined.

Discussion

In the present study, we demonstrate that SHP2 inhibition promotes anti-tumor immunity by

modulating both innate and adaptive immune cells. We propose that, although induction of anti-

tumor immunity by SHP2 inhibition is T-cell-dependent, a major driver of the response is

modulation of the macrophage compartment rather than a direct effect on T-cell signaling, thus

differentiating SHP2 inhibition from checkpoint blockade. Our data support a model in which

SHP2 inhibition has a direct impact on the viability of TAMs, thereby promoting a less

immunosuppressive tumor microenvironment. An appreciation of the tumor-extrinsic immune-

modulatory mechanisms of SHP2 should be instructive to the clinical evaluation of SHP2

allosteric inhibitors as a novel molecular therapeutic strategy in cancer patients.

Consistent with the proposed role of SHP2 as a downstream transducer of PD-1

checkpoint signaling in T cells (25,27-33,36,49) we have observed similarities between the in

vivo responses to SHP2 inhibition and immune checkpoint blockade in the tumor immune

microenvironment. We and others (9) have shown that an increase in tumor infiltrating CD8+T

cells is essential for SHP2 inhibitor-mediated control of established tumor growth and that these

T cells express less PD-1, suggesting that they are less exhausted in response to chronic antigen

exposure. However, while we have found a general concordance between the responses to anti-

PD-1 and SHP2 inhibition in various in vitro readouts of T-cell function, we have been unable to

demonstrate that pharmacological inhibition of SHP2 is equivalent to PD-1 blockade. In

particular, the disparate magnitude of the responses suggests that SHP2 is not the sole effector of

inhibitory PD-1 signaling in these model systems, as has been proposed previously (11,37). The

failure of RMC-4550 to phenocopy the effects of anti-PD-1 in the LCMV T-cell exhaustion

model in vivo also points to a greater complexity in PD-1 signaling than has perhaps been

appreciated thus far. In summary, while the present observations are consistent with a role for

SHP2 in PD-1 signal transduction and T-cell biology, the precise role for SHP2 in this pathway

vis a vis other redundant mechanisms, has yet to be elucidated.

More striking is the enhancement of tumor growth inhibition that we and others (9)

observe with the combination of global SHP2 inhibition and checkpoint blockade; this is

indicative of additional functions for SHP2 beyond checkpoint transduction in T-cells.

Significantly, we found that SHP2 inhibition had a profound impact on the survival and function

of suppressive monocytic immune cells such as TAMs and MDSCs. Here we demonstrate using

a pharmacological approach that SHP2 is a positive regulatory of ERK signaling downstream of

CSF-1R in human monocytes and murine BMDMs, which is in agreement with previous studies

using genetic deletion of PTPN11 (50). The inhibition of CSF-1R pro-survival signaling likely

accounts for the selective effects on M2 macrophage populations, as has been observed

previously with CSF-1R inhibitors (51-54), and is supported by the in vitro experiments in the

present study. The selective depletion of M2 macrophages in the tumor microenvironment after

SHP2 inhibition, without major effects on the M1 population, has important translational

implications. We did not observe effects of SHP2 inhibition on GM-CSF-differentiated




14

macrophages in vitro, suggesting that SHP2 does not play a role downstream of this receptor.

The GM-CSF receptor transduces pro-survival signals in M1 macrophages, which may be an

explanation of why SHP2 inhibition spares M1 cells and instead has a selective effect on M2s. In

addition to its role as a positive regulator of the RAS pathway, SHP2 has also been proposed to

negatively regulate STAT1 activation downstream of IFN signaling (55-57). As IFN/STAT1

signaling is important in M1 macrophage activation (58), inhibition of SHP2 may be supporting

a feed-forward loop for M1 macrophage polarization and survival, which encompasses not only

macrophage-intrinsic effects on signaling, but is influenced by the infiltration of IFN producing

CD8+ T-cells into the tumor. Consistent with this hypothesis, IFNγ or CD8+T cell depletion

induced an overall decrease in M1 frequency although a SHP2-inhibitor mediated increase was

still apparent. A role for SHP2 downstream of PD-1 in myeloid cells may also be possible. PD-1

signaling in myeloid cells can dampen anti-tumor immunity by regulating lineage fate

commitment and function of myeloid cells (59). Myeloid specific deletion of PD-1 in tumor

bearing mice resulted in a diminished accumulation of immature immunosuppressive cells and

an enhanced output of differentiated, inflammatory effector mMDSCs and phagocytic

macrophages, a phenotype similar to that of SHP2 inhibition.

Adaptive responses to signals in the tumor microenvironment are not restricted to the

immune compartment. There is compelling evidence that the infiltration of CD8+ T-cells can

induce production of CSF-1 by melanoma cells and other cancers by secretion of IFN- and

TNF-α (48,60), an effect we also observed in vitro in the colon CT26 model. Increased levels of

CSF-1 promote an increase in immunosuppressive M2 macrophages, via CSF-1R activation, and

a negative correlation with overall patient survival (48). The opposing effects of CD8+ T-cell

infiltration induced by checkpoint blockade could be counteracted by combination with anti-

CSF-1R therapies in a murine melanoma model (48). Intriguingly, our results suggest that SHP2

inhibition has the potential both to induce CD8+ T-cell infiltration and simultaneously to

counteract its negative consequences by suppressing CSF-1R signaling and therefore contract the

immunosuppressive macrophage population in the tumor microenvironment. This mechanism of

action may contribute to the enhanced anti-tumor activity we observed with RMC-4550 in

combination with checkpoint blockade. Correspondingly, it may account for the superior tumor

growth inhibition observed with the SHP2 inhibitor relative to anti-CSF-1R.

The potential for SHP2 inhibitors to provide therapeutic benefit in solid tumors bearing

SHP2-sensitive oncoproteins, in particular in non-small cell lung cancer (NSCLC), is the focus

of intensive clinical investigation. Multiple, rational combination strategies for a SHP2 inhibitor

with agents that target alternate nodes in the RAS-MAPK pathway (e.g. MEK (6); KRASG12C

(61) or RTK (62) inhibitors) or extra-proliferative functions of RAS (CDK4/6 (63)), have also

been proposed. The present data provide a strong rationale for a clinical combination strategy

with a SHP2 inhibitor and agents that target the immune system directly, such as anti-PD1 and

anti-CSF-1R. Patients bearing tumors that harbor oncogenic driver mutations sensitive to SHP2,

and with established clinical sensitivity to checkpoint inhibitors, for example KRASG12C

mutant-

NSCLC patients, could be particularly susceptible to this combination therapy. On the other hand

the present preclinical findings suggest that SHP2 inhibition seems unlikely to increase

sensitivity to an immune checkpoint inhibitor in checkpoint resistant tumors.

In summary, we have shown using preclinical models that SHP2 plays a central role in

inducing immune suppression in the tumor microenvironment both by inhibiting T-cells and

supporting the viability of pro-tumorigenic macrophages. SHP2 inhibition is an attractive




15

investigational therapeutic strategy with potential dual activity: targeted inhibition of RAS-

MAPK dependent tumor growth and liberation of anti-tumor immune responses by

transformation of the tumor microenvironment.

Acknowledgments. We 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 the

respective research teams at the following contract research organizations for the conduct of in

vitro and in vivo studies: MI Bioresearch (Ann Arbor, Michigan), HDB (San Diego, California),

WuXI Apptec (Shanghai, China), Ensigna (Brisbane, California), HistoTox Labs (Boulder,

Colorado) and PAIRimmune Inc (Québec, Canada). This work was supported in part by Sanofi,

Paris, France. The authors are full time employees of Revolution Medicines and declare no

competing financial interests.

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Figure Legends

Fig.1. SHP2 inhibition induces anti-tumor immunity in checkpoint blockade-sensitive

tumors and is additive in combination with a checkpoint inhibitor. (A-B) Oral daily

administration of 30 mg/kg RMC-4550 was used in all experiments. (A), Activity of RMC-4550

in A20, MC38 or CT26 tumor bearing immunocompetent mice. Kaplan-Meier plot showing

percentage of animals with tumor burden below 2000 mm3 for the duration of this study. (B),

Lack of activity of RMC-4550 in CT26 tumor bearing RAG-2 deficient immunocompromised

mice. (C), Activity of RMC-4550 in CT26 tumor bearing immunocompetent mice depleted of

CD4+ and CD8+T cells or CD8+T cells alone. Left two panels show tumor growth of individual

mice for each experimental group described, right panel shows Kaplan-Meier plot displaying

percentage of animals with tumor burden below 2000 mm3. (D), Activity of RMC-4550, anti-

PD-L1 (10 mg/kg, IP on days 3, 6, 10 and 13 post-implantation) or combination of both in CT26

tumor bearing immunocompetent mice. Dashed arrow indicates last day of treatment of RMC-

4550. Tumor growth of individual mice for each experimental group described and Kaplan–

Meier curves (right) are shown. (E) Same as D but using anti-CTLA4 (10 mg/kg, IP on days 7,

10, 14 and 17 post-implantation) as checkpoint inhibitor. Kaplan–Meier curves were compared

using the Mantel-Cox Log-Rank test (A-E, except for A20, used Logrank test for trend); n=10

animals per group (*P < 0.05; ***P < 0.001; ****P < 0.0001). TFS = Tumor-free survivors.

Fig. 2. SHP2 inhibition stimulates adaptive immunity in similar manner to checkpoint

inhibition. CT26 tumors derived from similar experiments as shown in Fig.1D or 1E, were taken

at day 16, after 13 or 9 days of treatment with anti-PD-L1 (A) or anti-CTLA4 (B, C, D)

respectively, and tumor immune cell infiltrates were analyzed by flow cytometry or

immunohistochemistry. (A, B), Quantification by flow cytometry of CD8+T or CD4+T cells in

CD45+TILs, ratio of CD8+T/Treg and PD-1 mean fluorescence intensity (MFI) of CD8+T cells.

(C), Quantification of CD8+T positive cells in CT26 tumors, as percentage of total number of

cells in each tumor section (graph in center) or in edge of tumor (right), determined by

immunohistochemistry. (D), Quantification by flow cytometry of MHC class I or PD-L1 MFI of

CD45 negative tumor cells and after in vivo depletion of IFN or CD8+T cells. (E), NFAT-

luciferase reporter gene PD-1/PD-L1 bioassay shows NFAT activation in response to increasing

concentrations of anti-PD-1 or RMC-4550. (F) SEB (top) or MLR (bottom) assay with human

cells from healthy donors shows secretion of IL-2 or IFN in response of increasing

concentrations of anti-PD-1 (1 ng/ml to 10 g/ml) or RMC-4550 (0.1 nM to 5 M). (A-C) Data

represent analysis of 5 mice per group, mean ± s.e.m. One-Way ANOVA followed by Holm-

Sidak test (A-B) or Tukey (C-D) (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Fig. 3. SHP2 inhibition modulates innate immunity, an effect not seen with checkpoint

blockade. CT26 tumors derived from similar experiments as shown in Fig.1D-E, were taken at

day 16, after 13 or 9 days of treatment with anti-PD-L1 (B, G) or anti-CTLA4 (A, C, D, H)

respectively, and tumor myeloid cell infiltrates were analyzed by flow cytometry or

immunohistochemistry. (A), Representative flow cytometric analysis of MHC class II and

CD206 expression of tumor associated macrophages (TAMs) defined as CD45+/CD3-/CD19-

/CD11b+/Ly6G-/Ly6C low/F4/80+. Gates indicate strategy to define M1 and M2 TAMs. (B-C)

Quantification by flow cytometry of CD11b+, F4/80+, M1 or M2 of CD45+ TILs in each




20

experimental group as indicated. (D), Immunohistochemistry analysis for F4/80+ cells in

consecutive sections of same tumors analyzed in Figure 2C. Quantification of F4/80+ 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 M1+ (top) or M2+ (bottom) tumor associated

macrophages upon in vivo depletion of IFN or CD8+T cells in CT26 tumors derived from the

same experiment as in Figure 2D. (F) MHC class I (MHCI) mean of fluorescence intensity

(MFI) analyzed by flow cytometry on M1 and M2 cells gated as in (E). (G, H), Quantification by

flow cytometry of mMDSC (CD45+/CD3-/CD19-/CD11b+/Ly-6C High/Ly-6G-) or gMDSC

(CD45+/CD3-/CD19-/CD11b+/Ly-6G+). (B-H) Data represent analysis of 5 mice per group,

mean ± s.e.m. One-Way ANOVA followed by Tukey (*P < 0.05; **P < 0.01; ***P < 0.001;

****P < 0.0001).

Fig. 4. SHP2 inhibition suppresses CSF-1R signaling and selectively affects viability of M2

macrophages. SHP2 inhibition exhibits greater anti-tumor activity relative to CSF-1R

inhibition in vivo. (A) The effect of RMC-4550 (72h) on proliferation of murine BM cells

grown in either CSF-1 (IC50 = 13 nM) or GM-CSF (IC50 not calculated due to shallow depth of

inhibition). (B) The effect of RMC-4550 (78h) on caspase 3/7 activation of murine BM cells. (C)

Effects of RMC-4550 on cellular pERK levels after acute stimulation of murine BM cells with

CSF-1 (IC50 = 3 nM) or GM-CSF (IC50 = 93 nM). (D) Western blot showing effects of RMC-

4550 and BLZ-945 on pERK levels of human monocytes acutely stimulated with CSF-1 for 5

minutes. (E) Murine BMDMs were polarized to M1 (IFN-, LPS) or M2 (IL-4) phenotypes for

24 h; Graph shows growth after teratment with RMC-4550 (72 h). (F) M1 and M2 polarized

BMDMs were treated with RMC-4550 (48h) and assayed for caspase 3/7 activation. (A-F) Data

represent the mean s.d. of n=2 or 3 independent biological experiments performed in technical

duplicate. (G-H), Activity of RMC-4550 (oral daily administration of 30 mg/kg), anti-CSF-1R (2

mg/mouse on staging day followed by 0.2 mg/mouse 6 days after staging) or combination of

both in CT26 tumor bearing immunocompetent mice. Tumor growth of individual mice for each

experimental group described (G) and Kaplan–Meier curves (H). Kaplan–Meier curves were

compared using the Mantel-Cox Log-Rank test; n=10 animals per group (*P < 0.05 **P < 0.01;

***P < 0.001; ****P < 0.0001).

Fig. 5. SHP2 inhibition is additive in combination with checkpoint inhibition in a SHP2

inhibitor-sensitive tumor model. (A-B), Activity of RMC-4550 (oral daily administration of 30

mg/kg), anti-PD-1 (10 mg/kg, IP every three days) or combination of both in EMT6 tumor

bearing immunocompetent mice. Tumor growth of individual mice for each experimental group

described (A) and Kaplan–Meier curves (B). Kaplan–Meier curves were compared using the

Mantel-Cox Log-Rank test; n=10 animals per group (*P < 0.05 **P < 0.01; ***P < 0.001; ****P

< 0.0001). TFS = Tumor-free survivors. (C), Working model for the effects of SHP2 inhibition

on anti-tumor immunity via modulation of both adaptive and innate mechanisms: blockade of

inhibitory signaling in CD8+T cells; direct and selective depletion of M2 pro-tumorigenic

macrophages through attenuation of CSF-1R signaling and induction of apoptotic cell death;

decrease of the suppressive potential of mMDSC; and increase of M1 macrophages via a

mechanism that is independent of CD8+T or IFN. Collectively these mechanisms contribute to

generate a less immunosuppressive environment and one that favors tumor cell elimination. In




21

those cancers with aberrant RAS/MAPK signaling, which are intrinsically sensitive to SHP2

inhibition, the ultimate impact on tumor cell growth will reflect integration of the both the direct,

targeted and anti-tumor immunity mechanisms.




Figure 1

A

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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 anti-tumor immunity by

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Allosteric inhibition of SHP2 stimulates anti-tumor …...2020/04/28 · Tumor Immunity by Transforming the Immunosuppressive Environment Elsa Quintana1, Christopher J. Schulze1,