stm.sciencemag.org/cgi/content/full/12/545/eaaz5387/DC1

Supplementary Materials for

Blocking immunosuppressive neutrophils deters pY696-EZH2–driven

brain metastases

Lin Zhang, Jun Yao, Yongkun Wei, Zhifen Zhou, Ping Li, Jingkun Qu, Akosua Badu-Nkansah, Xiangliang Yuan, Yu-Wen Huang, Kazutaka Fukumura, Xizeng Mao, Wei-Chao Chang, Jodi Saunus, Sunil Lakhani, Jason T. Huse,

Mien-Chie Hung, Dihua Yu*

*Corresponding author. Email: dyu@mdanderson.org

Published 27 May 2020, Sci. Transl. Med. 12, eaaz5387 (2020) DOI: 10.1126/scitranslmed.aaz5387

The PDF file includes:

Materials and Methods Fig. S1. EZH2 is highly expressed in brain metastases and promotes brain metastases in a methyltransferase-independent manner. Fig. S2. Nuclear Src binds to and phosphorylates EZH2 at Y696, reducing H3K27 trimethylation. Fig. S3. pY696-EZH2 drives brain metastasis with cytokine reprogramming but has little effect on cancer cell growth and invasion in vitro. Fig. S4. G-CSF recruits Arg1+/PD-L1+ neutrophils into the brains of mice bearing brain metastases. Fig. S5. c-Jun regulates CSF3 and IL1A expression. Fig. S6. pY696-EZH2 binds to and cooperates with RNA Pol II to up-regulate c-JUN transcription. Fig. S7. pY696-EZH2 is associated with pY416-Src, pS73-c-Jun, and Ki-67 staining in brain metastases of patients with breast cancer. Fig. S8. ICB combined with saracatinib impedes brain metastasis in mice. Fig. S9. Src-induced pY696-EZH2 interacts with RNA Pol II to recruit immunosuppressive neutrophils that enhance brain metastasis. References (80–83)

Other Supplementary Material for this manuscript includes the following: (available at stm.sciencemag.org/cgi/content/full/12/545/eaaz5387/DC1)

Data file S1 (Microsoft Excel format). Preprocessed A375 complementary DNA microarray data. Data file S2 (Microsoft Excel format). The reference table of cell lines and mouse groups.

Data file S3 (Microsoft Excel format). Original data for graphs. Data file S4 (Microsoft Excel format). Gene dataset results from ChIP sequencing analysis.

SUPPLEMENTARY MATERIALS

Materials and Methods

Reagents and cell culture

All common chemicals were purchased from Sigma. GSK126 was purchased from

Cayman Chemical Company (#15415) or Active Biochem Company (#A-1275); saracatinib

(#C43217) from AstraZeneca; SP600125 (#S1460) from Selleckchem; human G-CSF (#C002)

from Novoprotein; recombinant Src (#79635) from Abcam; and recombinant EZH2 (#50279)

from BPS Bioscience. Human G-CSF antibodies (#AB-214) and IgG control antibodies (#AB-

108) were purchased from R&D. Antibodies against EZH2 (#5246, #3147), Src (#2110, #2109),

pY416-Src (#6943), H3 (#4499), Arg1 (#93668), c-Jun (#9165), pS73-c-Jun (#9164), JNK

(#9258), and pT183/Y185-JNK (#4668) were purchased from Cell Signaling; antibodies for

EZH1 (#ab176115), Ki-67 (#ab15580), and CD8 (EPR21769) (#ab217344) from Abcam;

antibodies for SUZ12 (#MAB4184), EED (#AF5827), and S100A8 (#MAB3059) from R&D;

antibodies for H3K27me3 (#07-449) from EMD Millipore; antibodies against calgranulin A

(S100A8) (#sc-48352), Src (#sc-8056), lamin A/C (#sc-7292), lamin B (#sc-6217), and

granzyme B (2C5) (#sc-8022) from Santa Cruz; and RNA Pol II antibody (#NB200-598) from

Novus . Anti-mouse CD8α (#BE0061) and IgG control (#BE0090) antibodies, and antibodies for

PD-1 (#BEO146) and CTLA-4 (#BEO164) were from BioXcell. The pY696-EZH2 antibody was

designed and produced by contracting with LifeTein LLC. pY696-EZH2 peptide and EZH2

peptide were provided by LifeTein LLC. The DeadEND Colorimetric TUNEL System (#G7360)

and Dual-Luciferase Reporter Assay Kit (#E1910) were from Promega. LipoD293 DNA in vitro

Transfection Reagent (#SL100468) and pepMute siRNA Transfection Reagent (#SL100566)

were from SignaGen. The Myeloid-Derived Suppressor Cell Isolation Kit (#130-094-538) was

from Miltenyi Biotec. Human Cytokine Array Panel A (#ARY005) was from R&D. Mouse G-

CSF ELISA was from RayBiotech (ELM-GCSF-1). Q5 Site-Directed Mutagenesis Kit

(#E0554S) was from NEB. QIAprep Spin Miniprep Kit (#27104) and miRNeasy Mini Kit

(#217004) were from Qiagen. iScript cDNA Synthesis Kit (#1708890) was from Bio-Rad.

Cell lines

Human cancer cell lines (MCF7, HCC1954, and MDA-MB-231 breast cancer and A375

melanoma) and mouse cancer cell lines (4T1 and EMT6 mammary tumor) were purchased from

ATCC. B16BL6 mouse melanoma cells were provided by Dr. Isaiah Fidler’s lab. HEK 293FT

cells were purchased from Thermo Fisher Scientific. These cell lines were authenticated by the

Characterized Cell Line Core Facility at The University of Texas MD Anderson Cancer Center.

All cell lines were tested for and found to be free of mycoplasma contamination. Cancer cells

were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 supplemented with 10%

fetal bovine serum (FBS) (Thermo Fisher Scientific, #SH3007103).

RNA interference and cell line generation

For lentivirus production, a lentiviral expression plasmid and third-generation lentivirus

packaging vectors (psPAX2, pMD2.G) (#12260, #12259, Addgene) were used to transfect HEK

293FT cells using LipoD293 DNA in vitro Transfection Reagent (SignaGen). Lentiviruses were

collected 48 h after transfection for use in infecting cells. Infected cells were cultured under drug

selection to generate a stable cell line. Subcloned plasmids were used to transform DH5a-

competent Escherichia coli (#C737303, Thermo Fisher Scientific) or Stbl3-competent E. coli

(#18258012, Thermo Fisher Scientific). Transient transfection of siRNAs was performed using

pepMute siRNA Transfection Reagent (SignaGen). Lentiviral-based pLKO.1 shRNAs targeting

Src: sh#648 (TRCN0000038150), sh#1579 (TRCN0000038149); targeting CSF3: sh#441

(TRCN0000066008), sh#628 (TRCN0000066011); and targeting EZH2 sh#3

(TRCN0000040076), sh#4 (TRCN0000010475) were purchased from Sigma. c-Jun siRNAs:

si#61 (SASI_Hs02_00333461), si#78 (SASI_Hs02_00150278); EED siRNAs: #1

(SASI_Hs02_00336316), #2 (SASI_Hs02_00336317); and SUZ12 siRNAs: #1

(SASI_Hs01_00107270), #2 (SASI_Hs02_00347682) were purchased from Sigma. Src wt and

Src mutant (Y527F) were subcloned into pENTR3C vector then into pLOVE lentiviral

expression vector via Gateway cloning (Invitrogen). pGL4.10 (Luc.2) was purchased from

Promega. CSF3 Lentiviral Vector (Mouse) (pLenti-GIII-EF1a) was purchased from Applied

Biological Material, Inc. (Richmond). pLenti-HA-EZH2 lentiviral vectors were provided by Dr.

Mien-Chie Hung.

Site-directed mutagenesis and gene knockout

Site-directed mutagenesis was performed using a Q5 Site-Directed Mutagenesis Kit

(NEB, #E0554S). Y696 in EZH2 was replaced with either phenylalanine (F) or aspartic acid (D)

using the following primers:

Y696F: F5′-TAACCATCATAACTTTTGCAAAGCAGTTTGGATTTACCGAATGATTT-3′,

R5′-AAATCATTCGGTAAATCCAAACTGCTTTGCAAAAGTTATGGATGGTTA-3′,

Y696D: F5′-GTTAACCATCATAACTTTTGCATCGCAGTTTGGATTTACCGAATGAT-3′,

R5′-ATCATTCGGTAAATCCAAACTGCGATGCAAAAGTTATGATGGTTAAC-3′. H689 in

EZH2 was replaced with alanine (A) using the following primers: H689A: F5′-

CAAATGCTTCGGTAAATCCAAACTGC-3′, R5′-

CCGAAGCATTTGCAAAACGAATTTTG-3′.

To generate 4T1 and MDA-MB-231 EZH2-knockout cells, pSpCAs9 (BB)-2A-Puro

(PX459) V2 plasmids from Addgene were used following the protocol published by Ran et al.

(80). A single guide RNA (sgRNA) targeting EZH2 (sgRNA-EZH2) was designed using the

online CRISPR Design Tool (http://tools.genome-engineering.org): F5′-

CACCGTGGTGGATGCAACCCGCAA-3′, R5′-AAACTTGCGGGTTGCATCCACCAC-3′.

sgRNA-EZH2 oligos underwent annealing and then were inserted into a pSpCAs9 (BB)-2A-Puro

(PX459) V2 plasmid (#62988, Addgene) by Bbsl restriction enzyme digestion and T4 ligase-

mediated ligation. After ligation, plasmids were transformed into an Stbl3-competent E. coli

strain. The competent E. coli cells with PX459-sgRNA were plated on an ampicillin plate and

incubated overnight at 37 °C. Several clones were picked, and a single colony was inoculated in

LB media with ampicillin. After shaking overnight at 37 °C, plasmid DNA was isolated by using

a QIAprep Spin Miniprep Kit (Qiagen). Plasmid DNA was sequenced for validation of the

insertion by the Sequencing and Microarray Facility at MD Anderson. The sequence-verified

plasmids were transfected into cancer cells using Lipofectamine 2000. After puromycin

selection, DNA of stably transduced cancer cells was extracted for detection of DNA cleavage

with Surveyor PCR. After single cells were expanded into subclones, EZH2 protein expressions

were detected by Western blotting, and EZH2 DNA modifications were validated by DNA

sequencing. The 4T1.EZH2.KO cell line used for in vivo experiments (Fig. 1F-G, fig. S1E-F)

was a mixture pulled from the EZH2-knockout clones C41, C50, C52, C54, and C61. The

4T1.C50 clone was a subclone of EZH2-knockout 4T1, and the 231.3C13 clone was a subclone

of EZH2-knockout MDA-MB-231.

Western and dot blotting and IP

Western blotting and IP were performed as previously described (81). Briefly, cells were

lysed in lysis buffer (20 mM Tris at pH 7.0, 1% Triton X-100, 0.5% NP-40, 250 mM NaCl,

3 mM EDTA, and a protease inhibitor cocktail). Proteins were separated by sodium dodecyl

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose

membrane. The membranes were blocked with 5% milk for 30 min and then probed with

primary antibodies overnight at 4 °C. Next, the membranes were incubated with secondary

antibodies for 1 h at room temperature, and the bands were visualized with enhanced

chemiluminescence reagent (Thermo Fisher Scientific).

For IP, cells were washed twice with phosphate-buffered saline (PBS) and scraped into IP

lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris at pH 7.4, 1 mM EGTA, 1 mM

EDTA, 0.5 mM sodium orthovanadate, 0.4 mM phenylmethane sulfonyl fluoride [PMSF], and

0.5% NP-40). The total cell lysates were precleared by incubation with protein G-linked

agarose beads (#1124323300) for 2 h at 4 °C. After preclearing, lysates were incubated with the

primary antibody overnight at 4 °C and then with protein G-linked agarose beads for 1 h at 4 °C.

The beads were washed with IP buffer 3 times, and then the protein immunocomplex was

extracted from agarose and detected using SDS-PAGE and Western blotting.

We tested the specificity of an anti-pY696-EZH2 antibody that we generated via contract

with LifeTein, LLC by using dot blotting. We spotted onto a nitrocellulose membrane 1 mg/mL,

0.1 mg/mL, and 0.01 mg/mL (each in 2 μL dimethyl sulfoxide [DMSO] buffer) of pY696-EZH2

peptide (top 3 dots) or EZH2 peptide (bottom 3 dots) from LifeTein, LLC. After air drying and

blocking for 30 min with 5% milk, the membrane was incubated with a pY696-EZH2 antibody

(LifeTein, LLC) overnight at 4 °C, followed by washing and incubation with mouse secondary

antibodies for 1 h at room temperature and visualization with enhanced chemiluminescence

reagent.

Quantitative reverse transcription PCR

Briefly, total RNA was isolated using a miRNeasy Mini Kit (Qiagen) and then reverse-

transcribed using an iScript cDNA Synthesis Kit (Bio-Rad). Real-time PCR was then conducted

using Kapa Probe Fast Universal qPCR with SYBR Fast Universal qPCR Master Mix (Kapa

Biosystems) on a StepOnePlus real-time PCR system (Applied Biosystems). Relative mRNA

expression was quantified using the 2-ΔΔCt method with logarithmic transformation. SYBR

primers were obtained from Sigma or IDT. The TaqMan human EZH2 primer (#Hs00544833-

m1) was purchased from Life Technologies. The following primer sequences were used: SYBR

human EZH2: F5′-GGACTCAGAAGGCAGTGGAG-3′, R5′-GAGCTGTCTCAGTCGCATGT-

3′; mouse Ezh2: F5′-GTAGTAGACGCCACCCGCAAGGGTAACAAAATTGG-3′, R5′-

CCAATTTTGTTACCCTTGCGGGTGGCGTCTACTAC-3′; human c-JUN: F5′-

AAAGGATAGTGCGATGTTTC-3′, R5′-TAAAATCTGCCACCAATTCC-3′; mouse c-Jun:

F5′-AGCCTACCAACGTGAGTGCT-3′, R5′-AGAACGGTCCGTCACTTCAC-3′; mouse Csf3:

F5′-CAGGGAAGGAGATGGGTAAA-3′, R5′-GGAAGGGAGACCAGATGCT-3′; mouse Il1A:

F5′-CTCTAGAGCACCATGCTACAGAC-3′, R5′-TGGAATCCAGGGGAAACACTG-3′;

mouse SerpinE1: F5′-TTCAGCCCTTGCTTGCCTC-3′, R5′-

ACACTTTTACTCCGAAGTCGGT-3′; human ACTB: F5′-CATGTACGTTGCTATCCAGGC-

3′, R5′-CTCCTAATGTCACGCACGAT-3′; mouse Actb: F5′-AGTGTGACGTTGACATCCGT-

3′, R5′-TGCTAGGAGCCAGAGCAGTA-3′; human GAPDH: F5′-

CCATGAGAAGTATGACAACAGCC-3′, R5′-GGGTGCTAAGCAGTTGGTG-3′; mouse

Gapdh: F5′-AAGGTCATCCCAGAGCTGAA-3′, R5′-CTGCTTCACCACCTTCTTGA-3′.

Human IL1A (#HP 100200), CSF3 (#HP100107), and SERPINE1 (#HP100341) primers were

purchased from Sino Biological Company.

ChIP assay

ChIP assays were performed as previously described (82). Briefly, cells were fixed with

37% formaldehyde (final 0.5%), treated with glycine (final 125 mM), washed, resuspended in

ChIP lysis buffer (50 mM Tris at pH 8.1, 10 mM EDTA, 1% SDS, protease inhibitor cocktail,

and 10 mM PMSF), and then sonicated. The lysates containing soluble chromatin were incubated

with antibodies recognizing EZH2, H3K27me3, RNA Pol II, or IgG overnight at 4 °C, then

incubated for an additional 2 h at 4 °C with added protein G-linked agarose beads. The agarose

bead-bound complexes were then washed, and the protein-chromatin complexes were extracted

from the agarose beads with elution buffer. Reversal of the cross-linking of protein and DNA

was performed by incubating the elution with 10 mg/mL RNase A and 5 M NaCl overnight at 65

°C, followed by incubation with 0.5 M EDTA, 1 M Tris at pH 6.5, and 10 mg/mL proteinase K

for 1 h at 45 °C. Coprecipitated DNA was purified using a QIAquick spin column (Qiagen), and

2 μL DNA was analyzed with quantitative PCR using specific primers for the EZH2-binding

regions of the c-JUN promoter. The ChIP assay primers used were: ChIP-c-JUN_1F: 5′-

CCACGTTGAGAACACTCCGA-3′; ChIP-c-JUN_1R: 5′-CGAGTTTCGGATCGCCTACA-3′;

ChIP-c-JUN_2F: 5′-AGGGGTGGTTGTTGTTTCCC-3′; ChIP-c-JUN_2R: 5′-

GCCACCTTAAGGGCGGTATT-3′; ChIP-c-JUN_3F: 5′-CGCTAGCTCTGGGCAGTTAG-3′;

ChIP-c-JUN_3R: 5′-AGGCCTTGGGGTGACATCAT-3′; ChIP-c-JUN_4F: 5′-

CCAAGTTCAACAACCGGTGC-3′; ChIP-c-JUN_4R: 5′-GGAGACAAGTGGCAGAGTCC-3′;

ChIP-c-JUN_5F: 5′-TCCCGCACTCTTACTTGTCG-3′; ChIP-c-JUN_5R: 5′-

GTTGTTGAACTTGGGCGAGC-3′; HOXA9B_F: 5′-TCGCCAACCAAACACAACAGTC-3′;

HOXA9B_R: 5′-AAAGGGATCGTGCCGCTCTAC-3′. All fold enrichment values were

normalized to those of IgG. HOXA9B was used as a positive control for EZH2 and H3K27me3

binding and as a negative control for RNA Pol II binding.

ChIP-seq

ChIP-seq was performed in UTHealth CPRIT Cancer Genomics Core. The libraries were

prepared following the protocol of KAPA Hyper Prep Kit (KK8502, Roche Holding AG).

Briefly, the fragmented DNAs were repaired the both ends, and an adenine (A) was added to the

repaired ends. This end-repair and A-tailing procedure was performed at 20 0C for 30 minutes

followed by 65 0C for 30 minutes. Then, the adapters with unique indexes, which can identify

each sample during sequencing, were ligated to both ends of each fragmented DNA. After

ligation, the DNA fragments with adapters were cleaned up by magnet beads. The proper sizes of

DNA fragments were selected. The selected DNA fragments were quantified by quantitative

polymerase chain reaction (qPCR) and amplified by Bio-Rad C1000 Touch Thermal Cycler

(Bio-Rad Laboratories). The final library was qualified by Agilent Bioanalyzer 2100 (Agilent

Technologies) and quantified by qPCR. The libraries were pooled and subjected to a paired-end

75-cycle sequencing on an Illumina NextSeq 550 System (Illumina, Inc.) using High Output Kit

v2.5 (#20024907, Illumina, Inc.).

Luciferase reporter assay

The pGL4.10-c-JUN reporter (Promega) and a control Renilla luciferase vector were co-

transfected into tumor cell lines using Lipofectamine 2000 (Life Technologies). After 48 h,

luciferase activity was measured with a Dual-Luciferase Reporter Assay kit (Promega) on a

Luminometer 20/20 (Turner Biosystems). The c-JUN promoter (−130 bp to 170 bp) was

generated by amplification of a genomic DNA sequence by PCR using the designed primers and

then inserted upstream of the luciferase reporter gene in the pGL4.10 vector. The primer

sequence for the c-JUN_reporter_1 was F5′-CGGGGTACCCGGAGCATTACCTCATCCCG-3′,

R5′-CCGGATATCGACTATACTGCCGACCTGGC-3′.

Cytokine array

Cytokine Array Panels were used according to the manufacturer’s instructions (R&D

Systems, #ARY005). Briefly, cells were incubated for 48 h, then 1 mL of each cell culture

supernatant was collected. Each sample was diluted with array buffer, mixed with a cocktail of

biotinylated detection antibodies, and then incubated on Cytokine Array membrane at 4 °C

overnight. After washing, immunoreactivity was visualized with enhanced chemiluminescence

reagent. The intensity was measured using Image J software.

Saracatinib and SP600125 treatment in vitro

Cells were treated with 1 μM of the Src inhibitor saracatinib for 24 h or with 40 μM of

the JNK inhibitor SP600125 for 24 or 48 h, after which cell lysates were collected for Western

blot analysis.

Cytoplasmic and nuclear protein fractionation

Cell fractionation assays were performed as previously described (74). In brief, cells were

collected, resuspended in hypotonic buffer (10 mM HEPES at pH 8.0, 1.5 mM MgCl2, and 10

mM KCl), and incubated on ice for 15 min. They were then pelleted by centrifugation at 10,000

rpm at 4 °C for 5 min. The pellet was resuspended in lysis buffer (hypotonic buffer + 0.5% NP-

40) and incubated on ice for 30 min. Extracts were centrifuged at 13,000 rpm at 4 °C for 10 min,

and the supernatant was retained for the cytoplasmic fraction. The nuclear pellet was washed

twice with hypotonic buffer and then resuspended in lysis buffer (10 mM HEPES at pH 8.0, 1.5

mM MgCl2, 400 mM NaCl, 0.1 mM EDTA, and 20% glycerol), followed by incubation on ice

for 30 min. A 30-gauge syringe was used to break up the pellet and release the nuclear proteins.

Extracts were centrifuged at 13,000 rpm at 4 °C for 15 min, and the supernatant was retained as

the nuclear fraction.

Cell proliferation and soft agar assays

Cell proliferation was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) assay. One thousand cells were seeded per well (6 wells per

sample) in a 96-well plate, and cell growth was examined by staining with MTT agent (Thermo

Fisher Scientific, #M6496). The resulting intracellular purple formazan was solubilized using

DMSO and analyzed at optical densities of 570 to 620 nm using a Gen5 microplate reader

(BioTek, Inc.). Soft agar assays were performed as previously described (76). In brief, 0.5 mL of

0.6% agar in 1× culture medium was added to each well of a 24-well plate. After the bottom

layer of agar solidified, 0.5 mL of a mixture of 0.3% agar and 2000 cells in 1× culture medium

was added on top of the 0.6% agar. After 3 weeks, colonies were counted under a light

microscope.

Matrigel invasion and migration assays

For invasion assays, Transwell inserts were coated with 15% Matrigel for 2 h. Tumor

cells (50,000/well) in FBS-free medium were plated in the coated Transwell inserts. For

migration assays, tumor cells (10,000/well) in FBS-free medium were plated in uncoated

Transwell inserts. Medium containing 10% FBS was added to the lower compartment as a

chemical attractant. After culturing overnight or for up to 72 h, the invaded or migrated cells at

the bottom of the Transwell insert were stained with crystal violet and counted using a light

microscope.

Mass spectrometry

Liquid chromatography/tandem mass spectrometry (LC-MS/MS) was used to identify

EZH2 phosphorylation sites. MCF7 cell lysates were immunoprecipitated with an anti-EZH2

antibody. After protein gel electrophoresis, EZH2 bands were excised from the gels and

subjected to tryptic digestion. The phosphopeptides were isolated and enriched using

immobilized metal affinity chromatography and then analyzed with LC-MS/MS using an

Ultimate capillary LC system (LC Packings) coupled to a QSTARXL quadrupole time-of-flight

mass spectrometer (Applied Biosystems). The product ion spectra generated by the nanoscale

capillary LC-MS/MS were searched against National Center for Biotechnology Information

databases for exact matches using the ProID (Applied Biosystems) and MASCOT search

programs. Carbamidomethyl cysteine was set as a fixed modification, and serine, threonine, and

tyrosine phosphorylation were set as variable modifications. All phosphopeptides identified were

confirmed by manual interpretation of the spectra.

cDNA microarray

cDNA microarray analysis was performed by the Sequencing and Microarray Facility at

MD Anderson. Illumina bead arrays were used for mRNA profiling of RNA extracted from

A375 cells in culture, A375 subcutaneous tumors, and lung and brain metastases from mice

injected with A375 cells. mRNA expression was quantile-normalized, and MA plots were used

to verify the quality of replicate arrays.

In vitro kinase assay

MDA-MB-231 cells were transduced with Src WT plasmid (231.Src cells). After 48 h,

Src was immunoprecipitated from 231.Src cell extracts. Src isolated from 231.Src cells or 1 g of

recombinant Src (Abcam, #79635) was incubated with 1 µg of recombinant EZH2 (BPS

Bioscience, #50279) in kinase buffer supplemented with 5 µCi of [γ-32P]ATP and 50 mM cold

ATP for 30 min at 30 °C. Reaction products were resolved by SDS-PAGE, and 32P-labeled

EZH2 proteins were visualized by autoradiography.

GST-Src expression and purification

GST pulldown assays were performed according to standard protocols. Briefly, SRC

cDNA was cloned into the pGEX4T2 expression vector (Addgene, #27458101). A GST fusion

protein, GST-Src, was purified from E. coli using standard methods. GST-Src was incubated

with total cell lysates (500 µg) from MDA-MB-231 cells and glutathione sepharose 4B beads

(GE Healthcare) overnight at 4 °C. The beads were then washed, and the proteins were analyzed

using SDS-PAGE and Western blotting.

IHC, IF, and immunocytochemistry analyses

Standard IHC staining was performed as described previously (81). Briefly, after

deparaffinization and rehydration, 4-μm tissue sections were subjected to heat-induced epitope

retrieval. Slides were then incubated with various primary antibodies at 4 °C overnight after

blocking with serum-free protein block (#x0909, DAKO) for 10 min. Slides were then subjected

to color development with DAB or aminoethyl carbazole and hematoxylin counterstaining. Ten

visual fields from different areas of each tumor were independently evaluated by 2 pathologists

who were blinded to the experimental groups. Ki-67 and TUNEL staining in mouse tumors was

calculated as the percentage of positive cells per field and normalized to the total number of

cancer cells in each field. Necrotic areas in the tumors were excluded from evaluation. IHC

intensity scores were ranked into 4 groups: negative (−), positive-low (+), positive-medium (++),

and positive-high (+++). In IHC scoring of the tissue microarrays (TMAs) of the matched

primary breast tumor and brain metastasis patient samples, the score “low” corresponded to

“negative,” while the score “high” corresponded to the range from + to +++. “Ki-67 low” meant

that the percentage of positively stained tumor cells was less than 1%, while “Ki-67 high” meant

that the percentage of positively stained tumor cells was 1% or higher. Pathologists were blinded

to the group allocation during the staining and when assessing the outcomes.

IF staining was performed following a standard protocol as described previously (15).

Briefly, after antigen retrieval, slides were incubated with various primary antibodies at 4 °C

overnight. After washing, slides were incubated with fluorescent-labeled secondary antibodies,

washed again and mounted on coverslips with mounting medium containing DAPI. The

fluorescent signals were examined using a confocal microscope.

For IF staining of cells or immunocytochemistry, cells were seeded in 8-chamber culture

slides. After being washed twice with PBS, cells were fixed with 4% formaldehyde, and

permeabilized with 0.25% Triton X-100. The slides were blocked with serum-free protein block

(DAKO, #x0909), then incubated with a primary antibody overnight at 4 °C. Slides were then

subjected to color development with DAB, or incubated with fluorescent-labeled secondary

antibodies. DAB signals were examined using a light microscope, while the fluorescent signals

were examined using a confocal microscope.

For IF staining of blood cells, peripheral blood samples were collected from mice, and

the red cells were lysed. A drop of white cells was spread onto a glass slide. After air drying, the

slides were fixed with 100% methanol and permeabilized with 0.25% Triton X-100. The

subsequent steps are same as those in IF staining of cells.

Flow cytometry

Whole brain tissue was isolated from experimental mice and cut into small pieces using

scissors after removal of the meninges. The brain tissues were digested with 2 mg/mL

collagenase (Sigma, #C0130) for 30 min at 37 °C and then separated into single cells using a

tissue grinder and glass pestle. Single cells were washed twice in PBS, and then red blood cells

(RBCs) were removed using RBC lysis buffer (8.26 g NH4Cl, 1 g KHCO3, 0.037 g EDTA).

After centrifugation, the cell pellet was washed twice in PBS and suspended in magnetic-

activated cell sorting (MACS) buffer (1× PBS supplemented with 0.5% bovine serum albumin

and 2 mM EDTA). For flow cytometry, 1% or 2% of the whole brain cells were stained. Blood

samples were taken from the heart and collected into EDTA BD Microtainers (BD Biosciences).

After centrifugation, the serum was separated, and the RBCs were removed using RBC lysis

buffer. After another centrifugation, the cell pellet was washed twice in PBS and prepared for

staining. Cells were stained with Fixable Viability Dye (eFluor450; eBioscience, #65-0863-14,)

or CD45-APC-eFluor780 (30-F11, eBioscience, #47-0451-82), CD11b-BV510 (M1/70,

BioLegend, #101263), Gr1-PE/Cy7 (RB6-8C5, BioLegend, #108416), Gr1-PE (RB6-8C5,

BioLegend, #108407), Ly6G-Alexa-Fluor647 (1A8, BioLegend, #127610), Ly6C-FITC (H.K1.4,

BioLegend, #128005), F4/80-PE (BM8, BioLegend, #123110), CD8/PerCP-Cy5.5 (53-6.7,

BioLegend, #100734), CD4/PE (GK1.5, BioLegend, #100408), PD-L1-PE/Cy7 (10E.9G2,

BioLegend, #124314), or IgG2b k isotype-PE/Cy7 (RTK4530, BioLegend, #400618) antibodies.

Stained samples were analyzed using a BD FACSCanto II cytometer (BD Biosciences).

Identifying neutrophils from brain cells by FlowJo analysis involves multiple gating

steps. The first gating step was to distinguish the possible immune cell population from brain

cells on the basis of the cells’ forward scatter (FSC-A) and side scatter (SSC-A) properties.

Second, dead cells were excluded by positive staining with Fixable Viability Dye. Third, single

cells were identified by plotting their FSC-height (FSC-H) by FSC-area (FSC-A). The fourth

gating step was to identify immune cells by CD45+ staining. The fifth gating step was to identify

myeloid cells by CD11b+ CD45+ staining. Next, neutrophils were identified by CD45+ CD11b+

Gr1+ or CD45+ CD11b+ Ly6G+ staining.

Bone marrow-derived Ly6G+ cell isolation

Ly6G+ cells were isolated from the bone marrow of BALB/c or nude mice. The bone

marrow from femora and tibiae was flushed out with RPMI 1640 medium using a 10-mL

syringe with a 26G1/2 cannula. After centrifugation at 582 g for 10 min at room temperature,

the RBCs were lysed using RBC lysis buffer for 10 min at room temperature, then centrifuged

at 700g, 10 minutes. The cell pellet was suspended in ice-cold MACS buffer, then washed and

resuspended. Following the protocol of the Myeloid-Derived Suppressor Cell Isolation Kit

(Miltenyi Biotec, #130-094-538), the cells were labeled with anti-Ly-6G-biotin and anti-biotin

microbeads. The cell suspension was then loaded onto a MACS column, which was placed in the

magnetic field. The Gr1high Ly-6G+ cells were retained within the column and eluted as the

fraction whose biomarkers were verified by flow cytometry. These cells were cultured in RPMI

1640 with 2 mM L-glutamine, 10 mM HEPES, 20 µM 2-ME, 1% streptomycin/penicillin, and

10% FBS or 4T1-cell-conditioned medium.

Mice treatment

For GSK126 and vehicle treatments, the mice were assigned randomly to the treatment

groups. GSK126 was dissolved in 20% cyclodextrin (Captisol, CyDex Pharmaceuticals) and

adjusted to pH 4 to 4.5 with 1 N acetic acid following the instructions published by McCabe et

al. (30). GSK126 was administered via intraperitoneal injections 3 times per week at a dose of

150 mg/kg/mouse. Human G-CSF (20 μg/mouse) and IgG control (20 μg/mouse) antibodies

were intraperitoneally injected 3 times per week. Human G-CSF (4 μg/mouse) and PBS vehicle

were subcutaneously injected 3 times per week. These treatments were started at day 2 after

intracarotid or intracardiac injections.

For the CD8 T cell depletion experiment, female BALB/c mice were treated with

intraperitoneal injection of an initial dose of 200 μg/mouse of anti-CD8 (clone 2.43, #BE0061,

BioXCell) antibodies or IgG control (LTF-2, #BE0090, BioXCell) antibodies in PBS before

tumor cell injection (day −2), followed by similar dosing with 100 μg/mouse day 0, then every

5 days for the duration of the experiment. On day 0, mice were intracranially injected with

2000 4T1.C50.YD.Scr or 4T1.C50.YD.shCSF3 cells/mouse into the right hemisphere.

For the 4T1 combination treatment experiments, female BALB/c mice were intracranially

injected with 2000 4T1.Luc.GFP cells/mouse into the right hemisphere and with 20,000

4T1.Luc.GFP cells/mouse into the MFP. Mice were randomly assigned to treatment groups. All

treatments were started at day 3 post-injection. Saracatinib was prepared in vehicle (0.5%

hydroxypropyl-methylcellulose with 0.1% Tween 80) and administered by oral gavage (40

mg/kg) every day. ICB (anti-PD-1 antibody, 200 μg/mouse, and anti-CTLA-4 antibody, 100

μg/mouse) was administered by intraperitoneal injection every 3 days for a total of 5 times. For

the EMT6 combination treatment experiments, female BALB/c mice were intracranially injected

with 5000 EMT6 cells/mouse into the right hemisphere and with 50,000 EMT6 cells/mouse into

the MFP. Mice were randomly assigned to treatment groups. All treatments were started at day 6

post-injection. Saracatinib was administered by oral gavage (30 mg/kg) every day. ICB was

administered as for the 4T1 model.

Complete blood count

Complete blood counts, which measured the number and percentage of white blood cells

and RBCs in the blood, were performed by the Department of Veterinary Sciences at MD

Anderson.

RPPA

RPPA analysis of 231.3C13.pLenti, 231.3C13.WT, 231.3C13.YF, and 231.3C13.YD

cells was performed at the MD Anderson Functional Proteomics Reverse Phase Protein Array

Core Facility. Briefly, cellular proteins were denatured with 1% SDS, serially diluted, and

spotted on nitrocellulose-coated slides. Each slide was probed with a validated primary antibody

plus a biotin-conjugated secondary antibody. The signal obtained was amplified using a

DakoCytomation catalyzed system (Agilent Technologies) and visualized by a 3′,3′-

diaminobenzidine (DAB) colorimetric reaction. The slides were analyzed using customized

MicroVigene software (VigeneTech, Inc.). Each dilution curve was fitted with a logistic model

(“Super Curve Fitting,” developed at MD Anderson) (83) and normalized by median polish. The

intensity of the normalized log values for each antibody was compared among the 4 different cell

sublines using the GenePattern tool. Antibodies with higher signal expression in 231.3C13.WT

and 231.3C13.YD cells than in 231.3C13.pLenti and 231.3C13.YF cells were selected for

clustering and heatmapping.

RNA sequencing

RNA was extracted from frozen tissue with an AllPrep DNA/RNA/miRNA Universal Kit

(Qiagen), and the concentration was measured with a Qubit fluorometer (Thermo Fisher

Scientific). The RNA quality was evaluated with the 2200 TapeStation System (Agilent

Technologies). Total RNA was sent for mRNA sequencing to the Sequencing and Microarray

Facility at The University of Texas MD Anderson Cancer Center. The libraries were prepared

with a KAPA Stranded mRNA-Seq Kit and sequenced on Illumina HiSeq4000 platform using

the 75-bp paired-end option.

Fig. S1. EZH2 is highly expressed in brain metastases and promotes brain metastases in a

methyltransferase-independent manner. (A) Heat map showing the expression of 590

upregulated genes and 336 downregulated genes in experimental brain metastases induced by

intra-carotid artery injection of A375 human melanoma cells versus parental A375 cells in

culture, xenograft tumors induced by subcutaneous injection of A375 cells, and lung metastases

induced by tail vein injection of A375 cells. (B) EZH2 RNA expression in the different subtypes

of primary breast cancer using the dataset provided by Christina Curtis, et al. (29). (C) Heat map

showing expression of 1263 upregulated genes and 1811 downregulated genes in clinical brain

metastases versus lung and bone metastases from breast cancer patients (GSE14020, GPL570).

(D) 4T1 mouse mammary tumor cells were transfected with a pSpCAs9 (BB)-2A-Puro (PX459)

V2 plasmid, into which sgRNA-EZH2 oligos were inserted. Multiple subclones were selected

from different single cells. Loss of EZH2 protein expression in the indicated 4T1 subclones was

detected by Western blotting. The 4T1.EZH2.KO cell line used for in vivo experiments (Fig.

1E and F, fig. S1D) was a mixture pulled from the EZH2-knockout clones C41, C50, C52,

C54, and C61. The 4T1.C50 clone is the 4T1 subclone that had CRISPR/Cas9-mediated EZH2-

knockout and is the primary subclone used for subsequent studies. (E) Graph showing the fold

change in bioluminescence imaging (BLI) intensity in the head region of 3 subgroups of mice:

mice intracardially injected with 4T1.Ctrl cells and then treated with vehicle (4T1.Ctrl), mice

intracardially injected with 4T1.EZH2.KO cells and then treated with vehicle (4T1.EZH2.KO),

and mice intracardially injected with 4T1.Ctrl cells and then treated with GSK126 (4T1.GSK).

The BLI signal was normalized to the signal on the first day post-injection. Data are means ±

s.e.m. *P < 0.05, t-test. N.S., not significant. (F) Representative images of hematoxylin and

eosin (H&E) staining and immunohistochemistry (IHC) staining (H3K27me3, HOXA9, and

EZH2) and scores showing H3K27me3, Hoxa9, and EZH2 expression in brain metastases in the

3 subgroups (4T1.Ctrl, 4T1.EZH2.KO, and 4T1.GSK) described in (E). IHC score: −, negative;

+, low; ++, medium; +++ high staining, assessed by 2 pathologists. scale bar in H&E: 0.1 cm;

scale bar in IHC: 20 µm. (G) Western blotting detecting EZH2, H3K27me3, and H3 proteins in

4T1.C50 cells (EZH2 KO clone) transduced with empty vector (4T1.C50.pLenti), wild-type

EZH2 (4T1.C50.EZH2.WT), or a methyltransferase-defective EZH2 H689A mutant

(4T1.C50.EZH2.H689A).

Fig. S2. Nuclear Src binds to and phosphorylates EZH2 at Y696, reducing H3K27

trimethylation. (A) Amino acid sequence alignment of Y696-related regions of the EZH2

protein in the indicated species. Alignment was performed using BLAST. (B) Western blotting

showing GST-Src and EZH2 binding in vitro. The GST fusion protein GST-Src was purified

from E. coli pre-transduced with a pGEX4T2 plasmid and incubated with MDA-MB-231 cell

lysates and glutathione agarose beads. The beads were centrifuged, and the protein complexes

were detected by Western blotting using antibodies against EZH2 and Src. (C) Western blotting

detecting the indicated proteins in the cytoplasm and nuclear fractions of MDA-MB-231 and

HCC1954 cell lines. (D) Co-immunoprecipitation (IP) with antibodies against Src (top) and

EZH2 (bottom), followed by Western blotting (WB) of Src or EZH2 in nuclear fractions of 4T1

cells. Isotype-matched IgG served as control. (E) Dot blot assay detecting specific binding of

pY696-EZH2 antibody with Y696-phosphorylated EZH2 peptides but not non-phosphorylated

EZH2 peptides. Equal amounts of phospho- or non-phospho-EZH2 peptides (1:10 dilution from

the left to right dots) were immunoblotted with an anti-pY696-EZH2 antibody that we generated

via contracting with LifeTein, LLC. (F) Western blotting showing Src and β-actin proteins in

231.pLenti control cells and in 231.Src cells that were transduced with a Src-expressing vector.

(G) Western blotting of the indicated proteins in cell lysates of DMSO-treated 231.pLenti cells,

DMSO-treated 231.Src cells, and saracatinib-treated (1 μM) 231.Src cells (Sar.), 24 h post-

treatment. (H) Western blotting of the indicated proteins in cell lysates of DMSO-treated

231.pLenti cells, DMSO-treated 231.Src.Y527F cells, and saracatinib-treated (1 μM)

231.Src.Y527F cells (Sar.), 24 h post-treatment. (I) MDA-MB-231 cells were transduced with

sgRNA-EZH2-CRISPR-Cas9 plasmids, subclones from individual single cells of transduced

MDA-MB-231 cells were established, and then EZH2 and EZH1 expression in the indicated

subclones was detected by Western blotting. The 231.3C13 clone (circled) was the subclone of

EZH2-knockout MDA-MB-231 cells primarily used for subsequent studies. (J) The

CRISPR/Cas9-mediated EZH2-knockout 4T1.C50 cells were infected with control lentiviruses

(4T1.C50.pLenti), lentiviruses expressing wild-type EZH2 (4T1.C50.WT), lentiviruses

expressing EZH2 with Y696 mutated to phenylalanine (4T1.C50.YF), or lentiviruses expressing

EZH2 with Y696 mutated to aspartic acid (4T1.C50.YD). After establishment of stable sublines,

expression of the indicated proteins was detected by Western blotting. (K) Endogenous EZH2 in

MCF7 cells was knocked down by EZH2.shRNA, and then the cells were infected with control

lentiviruses (MCF7.shEZH2.pLenti), lentiviruses expressing wild-type EZH2

(MCF7.shEZH2.WT), lentiviruses expressing EZH2 with Y696 mutated to phenylalanine

(MCF7.shEZH2.YF), or lentiviruses expressing EZH2 with Y696 mutated to aspartic acid

(MCF7.shEZH2.YD). After establishment of stable sublines, expression of the indicated proteins

was detected by Western blotting.

Fig. S3. pY696-EZH2 drives brain metastasis with cytokine reprogramming but has little

effect on cancer cell growth and invasion in vitro. (A) Mice received intracarotid injections of

231.3C13.YD cells and were then treated with intraperitoneal (i.p) injections of vehicle (n = 8) or

GSK126 (n = 11) (150 mg/kg 3 times). Brain metastasis outgrowth was monitored by

bioluminescent imaging; quantification of the imaging signal (means ± s.e.m., t-test, N.S., not

significant) and representative images are shown. (B) Bone metastasis-free survival curves of

mice intracardially injected with 231.3C13.YF or 231.3C13.YD cells. Log-rank test, N.S. not

significant. (C) The tumor growth curve of mice injected with 231.3C13.YF or 231.3C13.YD

cells into mammary fat pad. Data are shown as means ± s.e.m, t-test, N.S. not significant. (D)

Cell proliferation was detected by MTT assays among 231.3C13.pLenti, 231.3C13.WT,

231.3C13.YF, and 231.3C13.YD cells. (E) Soft agar colony numbers among 231.3C13.pLenti,

231.3C13.WT, 231.3C13.YF, and 231.3C13.YD cells. (F) Representative images and

quantification showing invading or migrating MDA-MB-231, 231.3C13, 231.3C13.YF, and

231.3C13.YD cells. Scale bar: 100 µm. Data are means ± s.e.m., *P < 0.05, ** P < 0.01, N.S.

not significant, t-test. (G) qRT-PCR analysis of CSF3, IL1A, and SERPINE1 mRNA expression

in 231.3C13.pLenti, 231.3C13.WT, 231.3C13.YF, and 231.3C13.YD cells. Data are means ±

s.e.m. ***P < 0.001, ****P < 0.0001, t-test. (H) qRT-PCR analysis of Csf3, Il1A, and

SerpinE1 mRNA expressions in 4T1.C50.pLenti, 4T1.C50.WT, 4T1.C50.YF, and 4T1.C50.YD

cells. Data are means ± s.e.m. ****P < 0.0001, t-test. (I) qRT-PCR detection of CSF3 mRNA

expression in 4T1.C50.YD cells transduced with control shRNAs with a scrambled sequence

(sh.Scr) or with CSF3-targeting shRNAs (shCSF3 #441 and #681). Data are means ± s.e.m.

***P < 0.001, ****P < 0.0001, t-test. (J) qRT-PCR detection of CSF3 mRNA expression in

4T1.C50.YF cells transduced with lentiviruses harboring a control vector (4T1.C50.YF.vector)

or a CSF3-expressing construct (4T1.C50.YF.CSF3). Data are means ± s.e.m. ****P < 0.0001,

t-test.

Fig. S4. G-CSF recruits Arg1+/PD-L1+ neutrophils into the brains of mice bearing brain

metastases. (A) Gating scheme for isolating neutrophils by flow cytometry. (B) Representative

immunofluorescence staining (left) and quantification (right) of S100A8-positive neutrophils

in the brains of mice that were injected with 4T1.C50.YD cells transduced with sh.Scr

(4T1.C50.YD.sh.Scr) or shCSF3 #441 (4T1.C50.YD.shCSF3). Scale bar: 50 µm. Quantitative

data are means ± s.e.m. *P < 0.05, t-test. (C) Flow cytometry data showing the percentages

(left) and absolute numbers (right) of CD11b+ Ly6G+ cells infiltrating the brains of mice that

were injected with 4T1.C50.YD.sh.Scr and 4T1.C50.YD.shCSF3 cells. Absolute numbers are

shown as means ± s.e.m. *P < 0.05, t-test. (D) Flow cytometry data showing the absolute

numbers of CD11b+ F4/80+ cells infiltrating the brains of mice that were injected with

4T1.C50.YD.sh.Scr and 4T1.C50.YD.shCSF3 cells. Data are shown as means ± s.e.m. N.S. not

significant, t-test. (E) Representative immunofluorescence staining (left) and quantification

(right) of S100A8-positive cells in the brains of mice that were injected with

4T1.C50.YF.vector or 4T1.C50.YF.CSF3 cells. Scale bar: 50 µm. Quantitative data are means ±

s.e.m. ****P < 0.0001, t-test. (F) Flow cytometry data showing the percentages (left) and

absolute numbers (right) of CD11b+ Ly6G+ cells infiltrating the brains of mice that were

injected with 4T1.C50.YF.vector or 4T1.C50.YF.CSF3 cells. Absolute numbers are shown as

means ± s.e.m. **P < 0.01, t-test. (G) Quantification of Arg1-positive/S100A8-positive

neutrophils in the brains and in the peripheral blood from mice bearing brain metastases. Data

are means ± s.e.m. ****P < 0.0001, t-test. (H) Flow cytometry data showing PD-L1 expression

in neutrophils (CD11b+ Ly6G) isolated from the bone marrow of BALB/c mice and cultured for

4 days in RPMI 1640 medium (control) or in 4T1-cell-conditioned medium. (I) CFSE T cell

proliferation assay. CFSE-labeled murine splenocytes were cultured with no stimulus, with anti-

CD3/CD28 antibody (Ab, both 2 μg/ml), or with anti-CD3/CD28 Ab and 4T1- cell-conditioned

medium-cultured neutrophils. At day 3, cells were stained with PerCP-Cy5.5-conjugated anti-

CD8 Ab and flowJo cytometric histograms were shown CFSE dilution on CD8+ T cells. (J)

Bone marrow-derived neutrophils from nude mice were cultured in conditioned medium of

231.mCherry cells with G-CSF 20 ng/ml at Day 0; then 5000 231.mCherry cells were co-

cultured without or with 5X105 neutrophils in RPMI1640 with 10% FBS and G-CSF 20 ng/ml

for three days. Representative pictures of 231.mCherry cells co-cultured without or with

neutrophils under fluorescent microscope (left) and the quantification of 231.mCherry cell

numbers per view under fluorescent microscope (right, 10X). Scale bar: 100 µm. Quantitative

data are shown as means ± s.e.m. ****P

Fig. S5. c-Jun regulates CSF3 and IL1A expression. (A) Western blotting showing the

expressions of the indicated proteins in 4T1.C50.pLenti, 4T1.C50.WT, 4T1.C50.YF, and

4T1.C50.YD cells. (B, C) Western blotting of pS73-c-Jun protein (B) and qRT-PCR analysis of

CSF3 and IL1A mRNA expressions (C) in MDA-MB-231 cells treated with DMSO vehicle or

the JNK inhibitor SP600125 (SP, 40 μM, 24 h). Data are means ± s.e.m. **P < 0.01, ***P <

0.001, t-test. (D, E) Western blotting of pS73-c-Jun protein expressions (D) and qRT-PCR

analysis of Csf3 and Il1A mRNA expressions (E) in 4T1 cells treated with DMSO vehicle or

SP600125 (40 μM, 48 h). Data are means ± s.e.m. ***P < 0.001, t-test. (F) Western blotting of

pThr183/Tyr185-JNK and total JNK in 231.3C13.pLenti, 231.3C13.WT, 231.3C13.YF, and

231.3C13.YD cells. (G) Western blotting showing expressions of the indicated proteins in 4T1

cells treated with DMSO vehicle or saracatinib (sar.) (1 μM, 24 h). (H) Western blotting showing

EZH2 and Src protein expressions in MDA-MB-231 cells transduced with lentiviruses harboring

a control vector (231.pLenti) or wild-type Src (231.Src) co-expressing a control shRNA with a

scrambled sequence (231.Src.sh.Scr) or EZH2-targeting shRNAs (231.Src.shEZH2 #3 or

231.Src.shEZH2 #4). β-actin was used as a loading control.

Fig. S6. pY696-EZH2 binds to and cooperates with RNA Pol II to up-regulate c-JUN

transcription. (A) Western blotting showing the expressions of the indicated proteins in

231.3C13.pLenti, 231.3C13.WT, 231.3C13.YF, and 231.3C13.YD cells. (B) HEK 293FT cells

were transfected with control plasmids (pLenti), plasmids expressing wild-type EZH2 (WT),

plasmids expressing EZH2 with Y696 mutated to phenylalanine (YF), or plasmids expressing

EZH2 with Y696 mutated to aspartic acid (231.3C13.YD). EZH2 was immunoprecipitated from

the cell lysates of the HEK 293FT sublines, followed by detection of EED and SUZ12 by

Western blotting. (C) Western blotting showing the expresssions of the indicated proteins in

231.3C13.YD cells transduced with a control siRNA (siContl) or siRNAs targeting EED (siEED

#1 and siEED #2). (D) Western blotting showing the expressions of the indicated proteins in

231.3C13.YD cells transduced with siRNAs targeting SUZ12 (siSUZ #1 and siSUZ #2). (E)

Chromatin immunoprecipitation (ChIP)-qPCR analysis of EZH2 binding to c-JUN and HOXA9B

in 231.3C13.pLenti and wild-type EZH2-expressing 231.3C13.WT cells. Sonicated chromatin

was pulled down by antibodies against EZH2, followed by qPCR using the indicated primers. All

fold-enrichment values were normalized to the IgG value, which was defined as 1 and is

indicated by the red line. Data are means ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, t-test.

(F) ChIP-qPCR analysis of H3K27me3 binding to c-JUN and HOXA9B in 231.EZH2 cells.

Sonicated chromatin was pulled down by antibodies against H3K27me3, followed by qPCR

using the indicated primers. HOXA9B was used as a positive control, and all fold-enrichment

values were normalized to IgG values. Data are means ± s.e.m. **P < 0.01, t-test. (G) EZH2

binding to histone H3 substrate in 231.pLenti and 231.Src cells. Endogenous EZH2 was

immunoprecipitated from 231.pLenti and 231.Src cell lysates, followed by Western blotting with

H3 antibodies. (H) RNA Pol II binding to EZH2 in 231.pLenti and 231.Src cells. Endogenous

RNA Pol II was immunoprecipitated from 231.pLenti and 231.Src cell lysates, followed by

Western blotting with EZH2 antibodies.

Fig. S7. pY696-EZH2 is associated with pY416-Src, pS73-c-Jun, and Ki-67 staining in

brain metastases of patients with breast cancer. (A) Representative images of peptide-

competition immunohistochemistry (IHC) staining showing pY696-EZH2 antibody specificity.

MDA-MB-231 cells were stained with a pY696-EZH2 antibody (1:1000). The pY696-EZH2

antibody was used directly (upper right) or pre-incubated with phospho-EZH2 peptides (lower

left), non-phospho-EZH2 peptides (lower middle), or non-specific phospho-peptides (lower

right) at 37 °C for 2 h. (B-D) pY696-EZH2 staining was associated with pY416-Src staining

(B, P = 0.026, Fisher exact test), pS73-c-Jun staining (C, P = 0.037, Fisher exact test), and Ki-

67 staining (D, P = 0.0074, Fisher exact test) in breast cancer patients’ brain metastases. (E)

The overall survival (OS) and survival post brain metastasis (SPBM) in patients with high or low

expression of S100A8 in brain metastases. Log-rank test, **P

Fig. S8. ICB combined with saracatinib impedes brain metastasis in mice. (A) Secreted G-

CSF in the cultured supernatant of astrocytes, BV2 cells, and 4T1 tumor cells using ELISA.

Data are shown as means ± s.e.m. ****P < 0.0001, t-test. (B) Mammary fat pad (MFP) tumor

volumes of mice that were intracranially injected with 2 × 103 4T1.Luc.GFP cells and injected

in the MFP with 2 × 104 4T1.Luc.GFP cells. The mice were treated with vehicle, the Src

inhibitor saracatinib, immune checkpoint blockade (ICB), or saracatinib combined with ICB

(sar.+ICB). Tumor volumes were calculated based on caliper measurements of the major and

minor axes. Data are means ± standard errors of the mean (s.e.m.). *P < 0.05, ***P < 0.001, t-

test. (C) Kaplan-Meier curves showing overall survival of mice in the 4 groups described in

(B). **P < 0.01; ***P < 0.001, N.S., not significant, log-rank test. (D) Representative

immunohistochemistry (IHC) staining (S100A8, granzyme B [GzmB]) and

immunofluorescence staining (nucleus, CD8) in brain metastases of mice in the 4 groups

described in (B). Scale bar: 50 µm. (E) Representative IHC staining of Ki-67- and TUNEL-

positive cells in the brain metastases of mice in the 4 groups described in (B). Scale bar: 20 µm.

(F) Pictures showing the EMT6 MFP tumors in four groups treated with i) vehicle, ii) Src

inhibitor saracatinib (sar.), iii) ICB, or iv) sar+ICB. MFP tumors were collected at the time of

deaths. Scale bar: 1 cm.

Fig. S9. Src-induced pY696-EZH2 interacts with RNA Pol II to recruit immunosuppressive

neutrophils that enhance brain metastasis. Treatment with anti-G-CSF antibodies or immune

checkpoint blockade therapy (anti-PD-1 and anti-CTLA-4 antibodies) combined with the Src

inhibitor saracatinib can relieve immune suppression to inhibit brain metastasis outgrowth.

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