CANCER

A major chromatin regulatordetermines resistance of tumor cellsto T cell–mediated killingDeng Pan,1* Aya Kobayashi,1,2* Peng Jiang,3* Lucas Ferrari de Andrade,1 Rong En Tay,1

Adrienne M. Luoma,1 Daphne Tsoucas,3 Xintao Qiu,4 Klothilda Lim,4 Prakash Rao,4†Henry W. Long,4 Guo-Cheng Yuan,3 John Doench,5 Myles Brown,4

X. Shirley Liu,3‡ Kai W. Wucherpfennig1,6‡

Many human cancers are resistant to immunotherapy, for reasons that are poorlyunderstood. We used a genome-scale CRISPR-Cas9 screen to identify mechanismsof tumor cell resistance to killing by cytotoxic T cells, the central effectors ofantitumor immunity. Inactivation of >100 genes—including Pbrm1, Arid2, and Brd7,which encode components of the PBAF form of the SWI/SNF chromatin remodelingcomplex—sensitized mouse B16F10 melanoma cells to killing by T cells. Loss ofPBAF function increased tumor cell sensitivity to interferon-g, resulting in enhancedsecretion of chemokines that recruit effector T cells. Treatment-resistant tumorsbecame responsive to immunotherapy when Pbrm1 was inactivated. In many humancancers, expression of PBRM1 and ARID2 inversely correlated with expression ofT cell cytotoxicity genes, and Pbrm1-deficient murine melanomas were more stronglyinfiltrated by cytotoxic T cells.

Cancer immunotherapies that target inhib-itory receptors on T cells, including thePD-1 receptor, can induce durable responses,but most patients do not respond (1). Themechanisms that determine resistance to

these immunotherapies remain poorly under-stood. Cytotoxic T cells are key effectors of tumorimmunity on the basis of their ability to detectand kill transformed cells following T cell re-ceptor (TCR) recognition of peptide antigensbound to major histocompatibility complex (MHC)class I proteins (2). T cell–mediated cytotoxicitycan be especially efficient, but it is diminishedwhen MHC class I expression by tumor cells isreduced. Cytotoxicity is also inhibited when tu-mor cells express PD-L1, the ligand for the pro-grammed cell death 1 (PD-1) receptor on T cells(3). We hypothesized that sensitivity and resist-ance of tumor cells to T cell–mediated attack isdynamically regulated by multiple pathways in

tumor cells that could represent new targets forimmunotherapy.

Discovery of tumor cell–intrinsic genesregulating sensitivity and resistanceto T cell–mediated killing

Tumor cells transduced with a genome-scalegRNA library were subjected to selection withcytotoxic T cells to identify genes that controlledresistance to T cell–mediated killing (Fig. 1A).We selected the murine B16F10 melanoma cellline for this screen because it is resistant tocheckpoint blockade with antibodies targetingthe PD-1 and/or CTLA-4 (cytotoxic T lymphocyte–associated protein 4) receptors (4, 5). Inactivationof resistance genes resulted in depletion of thecorresponding gRNAs, but such depletion couldonly be detected with sufficient sensitivity whenmost tumor cells had sufficient Cas9 activity. Wetherefore selected a B16F10-Cas9 clone with highediting efficiency (fig. S1) and tested it with pos-itive controls that were either more resistant(B2m−/−) or sensitive (Cd274−/−) to T cell–mediatedcytotoxicity (fig. S2). This B16F10-Cas9 clone wasthen transduced with a genome-scale gRNA li-brary in a lentiviral vector (6). Selection was per-formed either with Pmel-1 T cells, which havea relatively low TCR affinity for an endogenousmelanoma antigen (7), or high-affinity OT-I T cells(8). Edited tumor cells were selected by 3-daycoculture with Pmel-1 CD8 T cells (or 1 day forOT-I T cells), and the representation of all gRNAswas quantified by Illumina sequencing of thegRNA cassette (Fig. 1A). The specificity of gRNAenrichment or depletion was demonstrated by

comparing selection with tumor-specific T cellsversus control T cells of irrelevant specificity(fig. S3). This comparison also controlled forpotential effects of gRNAs on cell proliferationand viability.A number of genes known to be essential for

T cell–mediated tumor immunity were identifiedamong the enriched gRNAs in both Pmel-1 andOT-I screens (Fig. 1B, fig. S4A, and tables S1and S2), including key genes in the MHC class Iand interferon-g (IFN-g) signaling pathways(9–11). Mutations in both MHC and interferonpathway genes were shown to confer resistanceto cancer immunotherapy (12, 13). T cell–basedCRISPR-Cas9 screens have been described bytwo other laboratories. One of these studies per-formed an in vivo screen covering 2368 murinegenes and highlighted the phosphatase Ptpn2as a new target for immunotherapy (14). Thesecond study focused on human tumor cells andT cells and reported that mutations in APLNRrender tumor cells resistant to T cell–mediatedcytotoxicity (15). Our approach emphasized sen-sitive detection of depleted gRNAs in a genome-wide manner, which allowed us to discoveradditional mechanisms conferring resistance toimmunotherapy.A notable result was that gRNAs were de-

pleted for a large number of genes (tables S1 andS2), indicating that inactivation of these genessensitized tumor cells to T cell–mediated killing.Top genes in this group included known neg-ative immune regulators, including Cd274 [encod-ing PD-L1 (16, 17)], Ptpn2 (18), and Serpinb9 (19)(Fig. 1C and fig. S4B). However, the vast major-ity of identified genes had not been previouslyimplicated in resistance to T cell–mediated killing(tables S1 and S2).

Pathways regulating resistance of tumorcells to T cell–mediated cytotoxicity

We performed gene set enrichment analysis toidentify known gene sets and pathways for genescorresponding to enriched or depleted gRNAs(tables S3 and S4). Five negative regulators oftheRas/MAPK (mitogen-activatedprotein kinase)pathway were identified among enriched gRNAs,includingNf1 (20),Dusp6 (21), Spred1 (22),Rasa2(23), and SPOP (24) (Fig. 1D). Ras pathway ac-tivation is very common among human cancersandmay not only promote tumor cell growth butalso attenuate tumor immunity. Braf is immedi-ately downstream of Ras, and small-moleculeinhibitors of mutant BRAFV600E elicit strongercytotoxic T cell responses in melanoma patientsand murine tumor models (25–27).Analysis of depleted gRNAs revealed a num-

ber of resistance pathways to T cell–mediatedkilling (Fig. 1, C and D, and table S4). All threeunique components of a SWI/SNF chromatinremodeling complex referred to as the polybromoand BRG1-associated factors (PBAF) complex(28, 29) were strongly depleted (Arid2, Pbrm1,and Brd7), providing strong evidence that thiscomplex conferred resistance to T cell–mediatedkilling (Fig. 1D). We also identified resistancegenes in the nuclear factor kB (NF-kB) pathway

RESEARCH

Pan et al., Science 359, 770–775 (2018) 16 February 2018 1 of 6

1Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02215, USA. 2AstellasPharma, Tokyo 103-8411, Japan. 3Department of Biostatisticsand Computational Biology, Dana-Farber Cancer Institute,Boston, MA 02215, USA. 4Department of Medical Oncology,Dana-Farber Cancer Institute, Boston, MA 02215, USA.5Genetic Perturbation Platform, Broad Institute of MIT andHarvard, Cambridge, MA 02142, USA. 6Department ofMicrobiology and Immunobiology, Harvard Medical School,Boston, MA 02115, USA.*These authors contributed equally to this work. †Present address:Harvard University Office of Technology Development, Cambridge,MA 02138, USA.‡Corresponding author. Email: kai_wucherpfennig@dfci.harvard.edu (K.W.W.); xsliu@jimmy.harvard.edu (X.S.L.)

on July 11, 2020

http://science.sciencemag.org/

Dow

nloaded from

Pan et al., Science 359, 770–775 (2018) 16 February 2018 2 of 6

Fig. 1. Systematic dis-covery of genes andpathways regulatingsensitivity andresistance of tumor cellsto Tcell–mediated killing.(A) Screening strategy.Cas9-expressing B16F10cells were transduced witha genome-scale gRNAlibrary (four gRNAs/gene).Edited B16F10 cells werecocultured with activatedcytotoxic T cells followedby Illumina sequencing ofgRNA representation.Specific selection wasperformed with Pmel-1T cells (specific for gp100melanoma antigen) orOT-I T cells (specific forOva peptide). Controlselection was performedwith T cells of irrelevantspecificity. (B) Top genesfor enriched gRNAsfrom Pmel-1 screen.Candidate genes wereplotted based on meanlog2 fold change of gRNAcounts compared to control selection and P values computed by MaGeCK (Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout).Dashed line indicates a FDR (false discovery rate) of 0.05. Annotated genes represent MHC class I (red), interferon (yellow), and Ras/MAPK(blue) pathways. (C) Top genes for depleted gRNAs from Pmel-1 screen. Genes related to the PBAF form of SWI/SNF complex (red), NF-kBpathway (blue), mTORC1 pathway (yellow), and known negative immune regulators (green) were annotated. (D) Selected pathways andcorresponding genes identified in the Pmel-1 screen. Color scale represents log2 fold change of average gRNA representation.

Pathways identified in the screen

0-4 4

LFC

Jak/S

tat pa

thway

Antige

n pre

senta

tion

Negati

ve re

gulat

ion of

MAPK

NF-κB pathw

ay

mTORC1 path

way

PBAF comple

x

Glycoly

sis

NAD meta

bolis

m

B16-Cas9

ControlT cells

Pmel-1/OT-IT cells

Resistant cells

Sensitive cells

gRNA libraryFDR=0.05

FDR=0.05

Genes with enriched gRNA

Genes with depleted gRNA

Log2 Fold Change

Log2 Fold Change

-Log

10 (

p va

lue)

-Log

10 (

p va

lue)

Jak1Stat1Ifngr2Ifngr1Jak2B2mH2-D1Tap1Tap2Nlrc5Dusp6Nf1Spred1SpopRasa2RelaIkbkbIkbkgRnf31OtulinSharpinCflarRragaRragcLamtor1Arid2Pbrm1Brd7Cd44Ero1lGaleGneNsdhlNamptNadk

ARID2 expression level

GZ

MB

/CD

8A r

atio

GZ

MB

/CD

8A r

atio

Frac

tion

0 500 1000 1500 2000

PBRM1 expression level

0 2000 4000 6000 8000

0 0

1 1

2 2

3 3

4 4

5 5cor=-0.26p=5.74e-9

ARID2 vs. GZMB/CD8A PBRM1 vs. GZMB/CD8A

Survival (months) Survival (months)0 0100 100200 200 300

0.2

00.

60.

40.

81.

0

0.2

00.

60.

40.

81.

0

CD8> 1 S.D. CD8< 1 S.D.

cor=-0.22p=1.265e-6

ARID2 low

ARID2 high

ARID2 low

ARID2 high

ARID2 vs. GZMB

BRCA

SARC

KIRCBLCA

THCA

-0.4 0.4-0.2 0.20Correlation

05

1015

20

-Log

10 (

p va

lue)

PBRM1 vs. GZMB

BRCA

BLCATHYM

THCA

OV

Correlation-0.4 0.4-0.2 0.20

02

46

8-L

og10

(p

valu

e)

Fig. 2. Expression of ARID2 and PBRM1 is negatively correlated withT cell cytotoxicity markers in TCGA data sets. (A) Correlation of ARID2and PBRM1 mRNA levels with GZMB mRNA levels in indicated cancers.Volcano plot showing the Spearman’s correlation and estimated significanceof ARID2 (left) or PBRM1 (right) with GZMB mRNA levels from RNA-seqdata across TCGA cancer types calculated by TIMER (Tumor ImmuneEstimation Resource) and adjusted for tumor purity (32). Each dot representsa cancer type in TCGA; red dots indicate significant correlations (P < 0.01).(B) Analysis of ARID2 and PBRM1 mRNA levels in relation to GZMB andCD8A as cytotoxicity and CD8 T cell infiltration markers, respectively.Spearman’s correlation of ARID2 (left) and PBRM1 (right) mRNA levels toGZMB/CD8A mRNA ratio in the TCGA melanoma data set. (C) Correlationof ARID2 expression level with survival of melanoma patients depending oncalculated level of CD8 T cell infiltration. All patients in the TCGA melanomastudy were divided according to the expression level of ARID2 (higher or lowerthan mean expression value of all patients). The impact of ARID2 expressionlevel on survival is shown for patients whose tumors had higher (>1 SD) orlower (<1 SD) expression of CD8 [(CD8A + CD8B)/2].

RESEARCH | RESEARCH ARTICLEon July 11, 2020

http://science.sciencemag.org/

Dow

nloaded from

(30) (Otulin, Rela, Ikbkg, Ikbkb, Rnf31, andSharpin) and key metabolic pathways, includ-ing mTORC1 (mechanistic target of rapamycincomplex 1) [Rraga, Rragc, and Lamtor1, whichare required for mTORC1 recruitment to lyso-somes (31)], glycolysis (including Nsdhl, Gne, Gale,Ero1l, and Cd44), and nicotinate/nicotinamidemetabolism (including Nadk and Nampt). TheNF-kB pathway was also identified as a resist-ance mechanism by Manguso et al. (14). Controlexperiments demonstrated that inactivation ofsuch genes did not merely increase sensitivityto cell death; inactivation of representative genes(Otulin, Dusp6, or Nf1) in B16F10-Cas9 cells didnot render them more sensitive to doxorubicin-induced cell death (fig. S5). Most of the iden-tified genes (253 of 313 genes) were validatedin a secondary screen (fig. S6), which also con-firmed the major pathways described above(fig. S7).

Clinical relevance of PBAF complex totumor immunity

We used TCGA RNA sequencing (RNA-seq) datasets and TIMER (32) to examine the relevanceof the CRIPSR screen (fig. S8) and PBAF com-plex in human cancers. We found that mRNAlevels of ARID2 and PBRM1 negatively correlated

with GZMB and PRF1 mRNA levels in many hu-man cancer types (Fig. 2A; fig. S9, A and B; andtable S5), suggesting that lower expression ofARID2 and PBRM1 is correlated with highercytotoxic activity contributed by CD8 T cells(fig. S9, C and D) in human cancers. This corre-lation was not merely explained by the degreeof CD8 T cell infiltration because ARID2 andPBRM1 mRNA levels were also negatively asso-ciated with the GZMB/CD8A ratio (Fig. 2B). Inaddition, we found that low ARID2 mRNA levelswere associated with a substantial survival ben-efit in melanoma patients, but only for thosetumors with a higher degree of infiltration byCD8 T cells (on the basis of CD8 expression)(Fig. 2C). These data suggest that ARID2 andPBRM1 affect tumor immunity in a variety ofhuman cancers.

Relevance of PBAF complex to immunecheckpoint blockade therapy

The SWI/SNF complex regulates chromatin ac-cessibility for transcription factors. The BAF ver-sion of SWI/SNF induces dissociation of Polycombrepressive complex 1 and 2 (PRC1 and PRC2) (33),but the PBAF complex may operate through adifferent biochemical mechanism. The two com-plexes share core subunits, but unique compo-

nents are ARID1A/B for the BAF (BRG1-associatedfactors) complex as well as ARID2, PBRM1, andBRD7 for the PBAF complex (Fig. 3A) (28). Tovalidate the role of the PBAF complex in regu-lating sensitivity to T cell–mediated killing, wegenerated B16F10 tumor cell lines in which thethree genes of the PBAF complex were individ-ually mutated by CRISPR-Cas9. Western blottingexperiments confirmed diminished amounts ofthe corresponding proteins in the mutant celllines (Fig. 3B). Inactivation of Arid2 diminishedprotein abundance of BRD7 and PBRM1, consistentwith a prior study (34), whereas inactivation ofPbrm1 did not affect protein abundance of ARID2or BRD7. Partial complexes with some chromatinremodeling activity may therefore remain in someof these knockout cell lines. Coculture of Arid2,Pbrm1, or Brd7 mutant tumor cells with cytotoxicT cells resulted in enhanced depletion of PBAFmutant cell lines compared to B16F10-Cas9 cellstransduced with a control gRNA (referred to ascontrol B16F10 tumor cells) in a 3-day cocultureassay (Fig. 3C). However, inactivation of Arid2,Pbrm1, or Brd7 genes did not alter cell prolife-ration over a 2-week period (fig. S10A).B16F10 tumor cells are resistant to checkpoint

blockade with antibodies against PD-1 (anti–PD-1)and/or anti–CTLA-4, and we therefore examined

Pan et al., Science 359, 770–775 (2018) 16 February 2018 3 of 6

Fig. 3. Inactivationof PBAF complexsensitizes tumorcells to T cell–mediated killingand synergizeswith checkpointblockade therapy.(A) Cartoonillustrating thecompositionof BAF and PBAFversions of SWI/SNF complex.(B) Western blotshowing proteinabundance ofARID2, BRD7,PBRM1, andGAPDH incontrol and indi-cated knockout celllines. (C) Greenfluorescent protein(GFP)–positiveArid2-, Pbrm1-, orBrd7-deficientB16F10 cells were mixed with GFP-negative control B16F10 cells atapproximately 1:1 ratio.Tumor cells were cocultured with Pmel-1 Tcells atindicated effector-to-target ratios for 3 days in triplicates; the fold changeof the percentage of GFP-positive tumor cells was determined by fluorescence-activated cell sorting.Two-way analysis of variance (ANOVA) was used todetermine statistical significance (****P < 0.0001). Values representmean ± SD. (D) Mice bearing control (n = 10) or Pbrm1-deficient B16F10tumors (n = 10) were treated with anti–PD-1 (a-PD-1, 200 mg/mouse) plusanti–CTLA-4 (a-CTLA-4, 100 mg/mouse), and tumor size was measured.Two-way ANOVAwas used to determine statistical significance for time points

when all mice were viable for tumor measurement. (E) Survival ofmice inoculated with control (n = 10) or Pbrm1-deficient B16F10 cells(n = 10) and treated with a-PD-1 plus a-CTLA-4. Log-rank (Mantel-Cox)test was used to determine statistical significance. (F) Flow cytometricanalysis of immune cell infiltration in Pbrm1-deficient and controlB16F10 tumors. The number of CD45+, CD4+, CD8+, and GranzymeB+ CD8+ Tcells was determined per gram of tumor. Mann-Whitneytest was used to determine significance (*P < 0.05, **P < 0.01). Valuesrepresent mean ± SD. Data in (C) to (F) are representative of twoindependent experiments.

Con

trol

gArid

2-1

gArid

2-2

gArid

2-3

gBrd

7-1

gBrd

7-2

gBrd

7-3

gPbr

m1-

1gP

brm

1-2

gPbr

m1-

3

ARID2

BRD7

PBRM1

GAPDH

BRG/BRM

BAF155 BAF170

BAF47

ARID1A/B

BAF60A/B/C

BAF57

DPF1/2/3

BAF complex

BAF53A/B

β-actinBRG ARID2

β-actin

BAF53A/B

PBAF complex

PBRM1

BRD7

BAF60A/B/C

BAF155 BAF170

BAF47

BAF57

DPF1/2/3

Contro

l

gCd2

74

gArid

2-1

gArid

2-2

gBrd

7-1

gBrd

7-2

gPbr

m1-

1

gPbr

m1-

20

0.5

1.0

1.5

Fol

d ch

ange

of

% G

FP

+ c

ells

No T cellsB16:Pmel=3:1B16:Pmel=1:1

****

****

****

****

****

****

****

0 5 10 15 20 25 30 35 40 45 500

50

100

Control α-PD-1/CTLA-4

Pbrm1-/- α-PD-1/CTLA-4

Days after tumor implantationDays after tumor implantation

Per

cent

age

surv

ival

Tum

or s

ize

(mm

2 )

**

Contro

l

Pbrm

1-/-

0

2

4

6

8

CD8+

Contro

l

Pbrm

1-/-

0

1

2

3

4*

CD45+

Cel

ls/ g

ram

of t

umor

( x

106 )

Cel

ls/ g

ram

of t

umor

( x

105 )

**

Contro

l

Pbrm

1-/-

0

0.5

1.0

1.5

CD4+

Cel

ls/ g

ram

of t

umor

( x

105 )

Contro

l

Pbrm

1-/-

0

1

2

3

4

5 *

Granzyme B+CD8+

Cel

ls/ g

ram

of t

umor

( x

104 )

Pbrm1-/- α-PD-1/CTLA-4

Control α-PD-1/CTLA-4 p=0.0005p=0.0001

0 5 10 15 20 25 300

200

400

600

RESEARCH | RESEARCH ARTICLEon July 11, 2020

http://science.sciencemag.org/

Dow

nloaded from

whether inactivation of Pbrm1 would renderB16F10 tumor cells sensitive to checkpoint blockade.Anti–PD-1 plus anti–CTLA-4 conferred therapeuticbenefit in mice bearing Pbrm1 mutant B16F10tumors, but this treatment was ineffective againstcontrol B16F10 tumors (Fig. 3, D and E, andfig. S10, B and C). Significantly increased num-bers of CD45+ immune cells, CD4 and CD8 T cells,and granzyme B+ CD8 T cells were present inPbrm1-deficient compared to control B16F10 tu-mors treated with PD-1 plus CTLA-4 checkpointblockade (Fig. 3F and fig. S10D). Single-cellRNA-seq analysis of sorted CD45+ immune cellsshowed that gene expression signatures associ-ated with productive antitumor immunity (IFN-gresponse, IFN-a response, and tumor necrosisfactor a signaling via NF-kB) were significantlyenriched in Pbrm1-deficient compared to controlB16F10 tumors for both myeloid cells (dendriticcells and M1-like macrophages) and lymphoidcells (T cells and natural killer cells) (fig. S11,A to C). These single-cell data also identifiedan increased percentage of dendritic cells and ahigher ratio of tumor-inhibitory M1-like macro-phages to tumor-promoting M2-like macrophagesin Pbrm1-deficient compared to control B16F10tumors (fig. S11D). Thus, inactivation of Pbrm1

not only sensitizes tumor cells to T cell–mediatedcytotoxicity but also results in a more favorabletumor microenvironment.

Regulation of IFN-g and mTORC1pathways by the PBAF complex

To investigate the molecular mechanisms bywhich the PBAF complex regulates the sensi-tivity of B16F10 tumor cells to T cell–mediatedkilling, we examined the transcriptome of PBAF-deficient B16F10 cells by RNA-seq. Arid2- andPbrm1-deficient B16F10 cells shared similar geneexpression profiles (fig. S12, A and B), consistentwith their critical role in the PBAF complex. Thetranscriptome of Brd7 mutant B16F10 cells wasmore distinct, suggesting that Brd7 may also havePBAF-independent functions (fig. S12A). mRNAsfor a number of metabolic pathways were con-cordantly down-regulated in Arid2 and Pbrm1mutant cells compared to control B16F10 tumorcells, in particular gene sets associated withmTORC1 activation and cholesterol homeostasis(fig. S12, C and D, and fig. S13). mTORC1 was alsoa major resistance pathway for T cell–mediatedcytotoxicity in the CRISPR-Cas9 screen (Fig. 1D).Silencing of BAF200 (Arid2) with a small inter-

fering RNA was shown to reduce the expression

of interferon induced transmembrane protein 1(IFITM1) by IFN-a but not other interferon-regulated genes (34). We systematically ex-amined whether the PBAF complex regulatesgene expression in response to IFN-g, giventhe importance of this T cell–derived cytokine fortumor immunity (12). RNA-seq analysis showedthat gene sets related to IFN-g and IFN-a re-sponse were significantly enriched among genesconcordantly up-regulated in Arid2- and Pbrm1-deficient cells compared to B16F10 control cellstreated with IFN-g (Fig. 4, A and B), suggestingthat ARID2 and PBRM1 suppressed the expressionof IFN-g–responsive genes. Many of the IFN-g–responsive genes suppressed by Arid2 and Pbrm1were relevant to innate immunity or encodedchemokines (Cxcl9 and Cxcl10) (Fig. 4C) (35).Pbrm1-deficient tumor cells also secreted sub-stantially larger amounts of CXCL9 and CXCL10—key chemokines for recruitment of effector T cellsthat express the CXCR3 chemokine receptor—compared to control B16F10 cells following IFN-gstimulation (Fig. 4, D to F) (35). Arid2-deficientcells had significantly increased surface levels ofH2-Kb over a range of IFN-g concentrations com-pared to control B16F10 cells. Also, all three mu-tants showed increased surface expression of

Pan et al., Science 359, 770–775 (2018) 16 February 2018 4 of 6

Fig. 4. Enhanced responsivenessto IFN-g stimulation by Arid2- andPbrm1-deficient tumor cells.(A to C) RNA-seq analysis of Arid2-or Pbrm1-deficient cells and controlB16F10 cells treated with IFN-g(10 ng/ml) for 24 hours. (A) Venndiagram showing differentiallyregulated mRNAs in the presence ofIFN-g. (B) Hallmark gene setsenriched for commonly up- or down-regulated mRNAs in both Arid2- andPbrm1-deficient cells compared tocontrol B16F10 cells in the presenceof IFN-g treatment [as shown in (A)].(C) Heat map showing expressionvalue (z-score based on cufflinkcount) of interferon-responsivegenes in control, Arid2-, and Pbrm1-deficient B16F10 cells following IFN-gtreatment. (D and E) Cxcl9 mRNAlevel (D) and Cxcl9 protein secretion(E) comparing Pbrm1-deficientand control B16F10 tumor cellsstimulated with IFN-g (10 ng/ml)for 24 hours. Values representmean ± SD. (F) Cxcl10 secretionby Pbrm1-deficient and controlB16F10 tumor cells stimulated withIFN-g (0, 0.5, and 1 ng/ml) for24 hours. Values representmean ± SD. One-way ANOVA (D andE) and two-way ANOVA (F) were usedto determine significance.**P < 0.01, ****P < 0.0001. Data in(D) and (F) are representative oftwo independent experiments.

Pbrm1-/-

Arid2-/-

Up-regulated genes

Pbrm1-/-

Arid2-/-

Down-regulated genes

Common up-regulated genes

Common down-regulated genes

0 5 10 15

TNF alpha signaling via NF-κBP53 pathway

GlycolysisEMT

IFN alpha responseIFN gamma response

-Log10 (FDR q-value)

0 5 10 15

Fatty acid metabolismAdipogenesis

HypoxiaEMT

mTORC1 signalingCholesterol homeostasis

-Log10 (FDR q-value)

Control Pbrm1-/- Arid2-/-

0 1 2-2 -1

Expression z-scores

Contro

l

gPbr

m1-

1

gPbr

m1-

20

2

4

6

8

Cxc

l9 m

RN

A (

rela

tive

to G

apdh

) ****

Contro

l

gPbr

m1-

1

gPbr

m1-

20

1000

2000

3000

CX

CL9

(pg

/ml)

****

****

0 0.5 10

500

1000

1500

IFNγ (ng/ml)C

XC

L10

(pg/

ml)

Control

gPbrm1-1

gPbrm1-2

****

****

****

****

Anti-viral response

T cell chemoattractant

RESEARCH | RESEARCH ARTICLEon July 11, 2020

http://science.sciencemag.org/

Dow

nloaded from

PD-L1 in response to IFN-g (fig. S14). Brd7- andPbrm1-deficient cells only showed enhanced sur-face expression of PD-L1 but not H2-Kb in re-sponse to IFN-g stimulation (fig. S14), which maybe due to partial complexes that retain someactivity. These data demonstrate that Arid2 andPbrm1 attenuate the responsiveness of B16F10 tu-mor cells to IFN-g, a key cytokine for the inter-action of tumor cells and T cells.

The PBAF complex regulates chromatinaccessibility of IFN-g−inducible genes

The major function of the SWI/SNF complex isto regulate chromatin accessibility for transcrip-tion factors. We therefore performed ATAC-seq(assay for transposase-accessible chromatin usingsequencing) to directly assess chromatin accessi-bility in Pbrm1-deficient and control B16F10 tu-mor cells with and without IFN-g treatment for24 hours. Following IFN-g treatment, a substan-tially larger number of genomic sites were acces-sible in Pbrm1-deficient than control B16F10 cells,consistent with the RNA-seq data (Fig. 5A). Sitesin cluster 1 (648 sites)weremore accessible beforeIFN-g treatment in Pbrm1-deficient compared tocontrol cells, suggesting that the correspondinggenes were poised to respond to IFN-g (Fig. 5,B and C, and fig. S15A). Also, 2708 sites (cluster III)showed enhanced accessibility following IFN-g

exposure in Pbrm1 mutant compared to controlB16F10 cells, but their accessibility was similarbetween the two cell lines in the absence of IFN-g(Fig. 5, B and C, and fig. S15B). Motif and targetgene prediction analysis suggests that these siteswere highly enriched with interferon regulatoryfactor (IRF) motifs and associated with IFN-regulated genes (fig. S15, C to E). Thus, inactivationof Pbrm1 enhances chromatin accessibility fortranscription factors at promoters or enhancersof many IFN-g-inducible genes.

Discussion

These data demonstrate that resistance to T cell–mediated cytotoxicity is regulated bymany genesand pathways in tumor cells. The correspondinggene products represent targets for immuno-therapy because inactivating mutations sensitizetumor cells to T cell–mediated attack. The inter-action between T cells and tumor cells is dy-namically regulated at many levels, includinginnate immune and metabolic pathways withintumor cells. The PBAF complex is of particularinterest because it reduces chromatin accessibil-ity for IFN-g–inducible genes within tumor cellsand thereby increases resistance toT cell–mediatedcytotoxicity.The PBAF complex is a tumor suppressor, and

inactivatingmutations in any of the three unique

genes of this complex (PBRM1,ARID2, andBRD7)are known to occur in a variety of human can-cers (28). For example, inactivating mutationsin PBRM1 are prevalent in clear cell renal cancer(~41% of patients) (36). A study byMiao et al. inthis issue demonstrates that PBRM1 mutationsin metastatic renal cancers are associated withimproved clinical responses toPD-1 orPD-L1 block-ade (37). Mutations in ARID2 and BRD7 are alsoobserved in a variety of other human cancers,including ARID2 mutations in melanoma (38).Human tumors with inactivating mutations inPBRM1,ARID2, andBRD7may therefore bemoresensitive to PD-1 blockade as well as other formsof immunotherapy in which cytotoxic T cellsserve as the main effector mechanism, includingcancer vaccines and adoptive T cell therapies.This study provides a mechanistic understand-ing for these clinical findings by demonstrat-ing that PBAF-deficient tumor cells are moresensitive to T cell–mediated cytotoxicity. We alsoshow that PBAF-deficient tumor cells producehigher amounts of chemokines (Cxcl9 and Cxcl10)in response to IFN-g, resulting in more efficientrecruitment of effector T cells into tumors (35).The immunotherapy field has thus far empha-

sized the targeting of inhibitory receptors expressedby immune cells. We propose that targeting oftumor cell–intrinsic resistance mechanisms toT cell–mediated cytotoxicity will be important toextend the benefit of immunotherapy to largerpatient populations, including those with cancersthat thus far are refractory to immunotherapy.

REFERENCES AND NOTES

1. P. Sharma, S. Hu-Lieskovan, J. A. Wargo, A. Ribas, Cell 168,707–723 (2017).

2. N. Zhang, M. J. Bevan, Immunity 35, 161–168 (2011).3. Y. Iwai et al., Proc. Natl. Acad. Sci. U.S.A. 99, 12293–12297 (2002).4. A. van Elsas, A. A. Hurwitz, J. P. Allison, J. Exp. Med. 190,

355–366 (1999).5. S. Chen et al., Cancer Immunol. Res. 3, 149–160 (2015).6. J. G. Doench et al., Nat. Biotechnol. 34, 184–191 (2016).7. W. W. Overwijk et al., J. Exp. Med. 198, 569–580 (2003).8. K. A. Hogquist et al., Cell 76, 17–27 (1994).9. J. S. Blum, P. A. Wearsch, P. Cresswell, Annu. Rev. Immunol.

31, 443–473 (2013).10. K. S. Kobayashi, P. J. van den Elsen, Nat. Rev. Immunol. 12,

813–820 (2012).11. B. S. Parker, J. Rautela, P. J. Hertzog, Nat. Rev. Cancer 16,

131–144 (2016).12. J. Gao et al., Cell 167, 397–404.e9 (2016).13. J. M. Zaretsky et al., N. Engl. J. Med. 375, 819–829 (2016).14. R. T. Manguso et al., Nature 547, 413–418 (2017).15. S. J. Patel et al., Nature 548, 537–542 (2017).16. H. Dong, G. Zhu, K. Tamada, L. Chen, Nat. Med. 5, 1365–1369

(1999).17. G. J. Freeman et al., J. Exp. Med. 192, 1027–1034 (2000).18. M. Kleppe et al., Blood 117, 7090–7098 (2011).19. D. Kaiserman, P. I. Bird, Cell Death Differ. 17, 586–595

(2010).20. N. Ratner, S. J. Miller, Nat. Rev. Cancer 15, 290–301 (2015).21. S. Messina et al., Oncogene 30, 3813–3820 (2011).22. T. N. Phoenix, S. Temple, Genes Dev. 24, 45–56 (2010).23. R. Arafeh et al., Nat. Genet. 47, 1408–1410 (2015).24. G. Li et al., Cancer Cell 25, 455–468 (2014).25. D. T. Frederick et al., Clin. Cancer Res. 19, 1225–1231 (2013).26. P. J. R. Ebert et al., Immunity 44, 609–621 (2016).27. R. C. Koya et al., Cancer Res. 72, 3928–3937 (2012).28. C. Kadoch, G. R. Crabtree, Sci. Adv. 1, e1500447 (2015).29. B. Lemon, C. Inouye, D. S. King, R. Tjian, Nature 414, 924–928

(2001).30. Q. Zhang, M. J. Lenardo, D. Baltimore, Cell 168, 37–57

(2017).31. Y. Sancak et al., Cell 141, 290–303 (2010).

Pan et al., Science 359, 770–775 (2018) 16 February 2018 5 of 6

Clu

ster

I si

tes

Clu

ster

III s

ites

Control Control + IFNγ Pbrm1-/- Pbrm1-/- + IFNγ1.8

0.2

1.8

0.2

Num

ber

of d

iffer

entia

l site

sThree clusters of differentially accessible sites

Cluster I(648 sites)

Cluster III(2708 sites)

Cluster II(3067 sites)

Control

Pbrm1

-/-0

2000

4000

6000

More accessible sitesLess accessible sites

3715

1677

5775

1766

Control Pbrm1-/-

Summary of differentially accessible sites (|LFC|>0.5)

Fig. 5. Enhanced chromatin accessibility for IFN-g–responsive genes in Pbrm1-deficient tumorcells. ATAC-seq was performed on Pbrm1-deficient and control B16F10 cells with or withoutIFN-g stimulation (10 ng/ml) for 24 hours. (A) Genome-wide analysis of differentially accessiblechromatin sites (|log2 fold change| > 0.5) following IFN-g stimulation in control versus Pbrm1-deficient B16F10 tumor cells. (B) Venn diagram illustrating accessible sites gained following IFN-gtreatment in control (blue) and Pbrm1-deficient (red) cells. (C) Chromatin accessibility heat maps forall sites in clusters I (top panel) and III (bottom panel). Aggregated reads within 2 kb of center ofdifferentially accessible regions are shown above heat maps.

RESEARCH | RESEARCH ARTICLEon July 11, 2020

http://science.sciencemag.org/

Dow

nloaded from

32. B. Li et al., Genome Biol. 17, 174 (2016).33. C. Kadoch et al., Nat. Genet. 49, 213–222 (2017).34. Z. Yan et al., Genes Dev. 19, 1662–1667 (2005).35. J. R. Groom, A. D. Luster, Immunol. Cell Biol. 89, 207–215

(2011).36. I. Varela et al., Nature 469, 539–542 (2011).37. D. Miao et al., Science 359, 801–806 (2018).38. E. Hodis et al., Cell 150, 251–263 (2012).

ACKNOWLEDGMENTS

We thank the staff of the Center for Cancer Genome Discovery andthe Molecular Biology Core Facility at Dana-Farber Cancer Institute(DFCI) for DNA sequencing. D.P. is a Cancer Research Institute–Robertson Foundation Fellow; L.F.d.A. was funded by a Friends

for Life Neuroblastoma Fellowship, and R.E.T. was supportedby an A*STAR Graduate Fellowship. A.M.L. is a Research Fellowof the Training Program in Cancer Immunology at DFCI (NIHgrant 1T32CA207021-01). This work was supported by aTransformative R01 grant from the National Cancer Institute, NIH(R01CA173750) (K.W.W.) and NIH (U24CA224316) (X.S.L.), andby a sponsored research agreement with Astellas Pharma. X.S.L. is acofounder of and is on the scientific advisory board of GV20Oncotherapy, a precision cancer medicine company. X.S.L. isalso on the scientific advisory board of 3DMedCare and is a paidconsultant for Genentech. J.D. is a paid consultant for TangoTherapeutics. K.W.W. and D.P. are inventors on patent applicationDFS-190.6 submitted by DFCI, which covers new targets forcancer immunotherapy. RNA-seq data have been deposited

at the Gene Expression Omnibus under accession numberGSE107670.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/359/6377/770/suppl/DC1Materials and MethodsFigs. S1 to S15Tables S1 to S5References (39–50)

21 June 2017; resubmitted 30 October 2017Accepted 14 December 2017Published online 4 January 201810.1126/science.aao1710

Pan et al., Science 359, 770–775 (2018) 16 February 2018 6 of 6

RESEARCH | RESEARCH ARTICLEon July 11, 2020

http://science.sciencemag.org/

Dow

nloaded from

mediated killing−A major chromatin regulator determines resistance of tumor cells to T cell

W. WucherpfennigXintao Qiu, Klothilda Lim, Prakash Rao, Henry W. Long, Guo-Cheng Yuan, John Doench, Myles Brown, X. Shirley Liu and Kai Deng Pan, Aya Kobayashi, Peng Jiang, Lucas Ferrari de Andrade, Rong En Tay, Adrienne M. Luoma, Daphne Tsoucas,

originally published online January 4, 2018DOI: 10.1126/science.aao1710 (6377), 770-775.359Science 

, this issue p. 770, p. 801; see also p. 745Sciencewhich encodes a subunit of the same SWI/SNF complex, were more likely to respond to the drugs.

,PBRM1carcinoma who had been treated with immune checkpoint inhibitors. Tumors harboring inactivating mutations in examined the genomic features of tumors from patients with metastatic renal cellet al.antitumor immunity. Miao

, leading to increased secretion of cytokines that promoteγmediated killing. The cells were more responsive to interferon-−specific SWI/SNF chromatin remodeling complex had been experimentally inactivated were more sensitive to T cell

discovered that tumor cells in which a et al.patients who benefit (see the Perspective by Ghorani and Quezada). Pan Understanding the mechanisms that determine tumor sensitivity to these drugs could potentially expand the number of

Immune checkpoint inhibitors induce durable tumor regressions in some, but not all, cancer patients.SNF'ing out antitumor immunity

ARTICLE TOOLS http://science.sciencemag.org/content/359/6377/770

MATERIALSSUPPLEMENTARY http://science.sciencemag.org/content/suppl/2018/01/03/science.aao1710.DC1

CONTENTRELATED

http://stm.sciencemag.org/content/scitransmed/10/422/eaao1641.fullhttp://stm.sciencemag.org/content/scitransmed/10/422/eaam7577.fullhttp://stm.sciencemag.org/content/scitransmed/9/415/eaan0401.fullhttp://stm.sciencemag.org/content/scitransmed/10/424/eaan5488.fullhttp://science.sciencemag.org/content/sci/359/6377/745.fullhttp://science.sciencemag.org/content/sci/359/6377/801.full

REFERENCES

http://science.sciencemag.org/content/359/6377/770#BIBLThis article cites 50 articles, 13 of which you can access for free

PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions

Terms of ServiceUse of this article is subject to the

is a registered trademark of AAAS.ScienceScience, 1200 New York Avenue NW, Washington, DC 20005. The title (print ISSN 0036-8075; online ISSN 1095-9203) is published by the American Association for the Advancement ofScience

Science. No claim to original U.S. Government WorksCopyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of

on July 11, 2020

http://science.sciencemag.org/

Dow

nloaded from