Genome-wide Screens Identify Lineage- and Tumor Specific- Modulating MHC-I and MHC-II Immunosurveillance in Human Lymphomas

Devin Dersh, James D. Phelan, Megan E. Gumina, Boya Wang, Jesse H. Arbuckle, Jaroslav Holly, Rigel J. Kishton, Tovah E. Markowitz, Mina O. Seedhom, Nathan Fridlyand, George W. Wright, Da Wei Huang, Michele Ceribelli, Craig J. Thomas, Justin B. Lack, Nicholas P. Restifo, Thomas M. Kristie, Louis M. Staudt, Jonathan W. Yewdell

Supplemental Figures

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9 9 9 8 9 9 8 9 8 8 8 9 8 8 9 8 9 9 n o 01 01 01 01 01 01 01 01 gu 01 01 01 01 01 01 01 01 01 01 i 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 te / / / / / / / / ct / / / / / / / / / / a 1 1 1 2 1 2 2 1 e 2 2 2 3 2 2 3 2 3 3 l D 0 0 0 1 0 0 1 0 l 1 1 1 0 1 1 0 1 0 0 mb / / / / / / / / / / / / / / / / / / a Co 6 6 6 8 6 6 8 3 8 8

28 28 28 19 28 28 19 28 d e s n L e 2 d C L 2 n 2 C C 4 L 2 co 6 s

2 FS Y1 Y3 Y8 H 8 HL4 HL5 a 1 T 9 L L L UD U 11 H I I I D L D D 1 1 rp VA C C C C I 293 O C U U a M Y MG Cell line BJAB D HB HLY1 K L M O O O R S S S T U WS WS I *Karpas422 HLA-C HLA-B *OCILY8 allele 1 - 07:02:01:17N or 07:02:01G allele 2 - 07:02:01G or 07:02:01:17N *SUDHL5 HLA-C allele 1 - 51:01:01G or 53:01:13 allele 2 - 58:01:01G or 58:08:02 HLA-B *WSUFSCCL allele 1 - 07:02:01G or 07:02:01:17N allele 2 - 12:02:01G or allele 1 - 27:127 or 27:05:02G allele 2 - 47:04 or 44:02:01G Supplemental Figure 1. HLA typing of DLBCL tumor lines. HLA typing of MHC-I and II loci of 18 DLBCLs commonly used throughout this work. NP, allele not present. Ambiguities listed at bottom. A B BJAB HBL1 3 p < 0.0001 p < 0.0001 1.5

1.5 2 1.0

7 1 0.5 1.0 validation: MHC-I remaining validation: MHC-I remaining

-4 -2 0 2 4 -4 -2 0 2 screen: seg score screen: seg score HLA-A2 / CD14 0.5 TMD8 SUDHL5 y = 0.9449x + 0.05649 p < 0.0001 p < 0.0001 R2 = 0.824 2 0.0 2 0.0 0.5 1.0 1.5

pan MHC-I / CD147 1 1 validation: MHC-I remaining validation: MHC-I remaining

-4 -2 0 2 -4 -2 0 2 screen: seg score screen: seg score

C HBL1 positive regulators BJAB positive regulators SUDHL5 positive regulators TMD8 positive regulators p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001

1.5 2.0

1.0 1.0 1.5 1.0 1.0 0.5 0.5 0.5 0.5

validation: MHC-I remaining 0.0 validation: MHC-I remaining 0.0 validation: MHC-I remaining 0.0 validation: MHC-I remaining 0.0 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 screen: -log(p-value) screen: -log(p-value) screen: -log(p-value) screen: -log(p-value)

HBL1 negative regulators BJAB negative regulators SUDHL5 negative regulators TMD8 negative regulators p = 0.0051 p = 0.3005 p = 0.0002 p = 0.0190 2.0 3.0 2.5 2.0

2.0 1.5 1.5 2.0 1.5 1.0 1.0 1.0 1.0 0.5 0.5 0.5 validation: MHC-I remaining validation: MHC-I remaining validation: MHC-I remaining 0.0 validation: MHC-I remaining 0.0 0.0 0.0 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0 1 2 3 4 5 screen: -log(p-value) screen: -log(p-value) screen: -log(p-value) screen: -log(p-value)

Supplemental Figure 2. HLA-A2 correlation with pan-MHC-I measurements and screen correlation with KO cells. (A) HLA-A2 per CD147 measurements were plotted against W6/32 per CD147 staining across a number of HBL1 KO cells; linear regression is plotted. (B) Individual KO cells were measured for surface MHC-I/CD147 and plotted against the CRISPR screen segregation score for the same . p-values represent likelihood of non-zero slope. (C) Surface MHC-I levels from individual gene KO cell lines during the validations were plotted against the -log(p-value) of the same gene from STARS analysis of the CRISPR screens. p-values represent likelihood of a non-zero slope. Only genes that were validated and received a score in STARS are plotted. 1.5 HBL1 g

1.0

0.5 fraction MHC-I remainin

0.0 1 1 T2 A1 ID2 FLII GPI P AF8 AP1 ZFX HTT TFG RFK B2M EED SPI1 IRF8 HGS IRF2 SPIB TA T JAK1 PPIG F8A1 T UFL1 TYK2 LCP1 AKT1 RFX5 BAP1 PFN1 CD70 TSC2 TSC1 EZH2 TCH1 TCH8 OAZ1 UBA5 USP1 APBP CALR GPS2 RER1 RCE1 LIPT1 ACTB CYLD EIF3L PDCL EDC4 BRD9 XPO6 SOD1 GRB2 BRAP XPOT PREP KDM8 SPOP YRM5 ANCB TYMS EIF3K CHUK SUOX BBIP1 SMG7 SIAH2 HIPK1 BCOR HLA-B RABIF DOHH S CHIC2 TP5F1 EP300 MOGS TTC14 LRP10 TXNL1 ZBTB2 SPPL3 PEX13 AP2A1 NAA25 RAB10 TRAF3 CSTF2 RAB35 RAB14 PTBP1 LSM12 T CDK13 SRSF6 DDX23 STUB1 CTBP1 KEAP1 L NLRC5 ERAP1 SURF4 RBBP7 AP1M1 LNPEP RAB7A AM98B UBE3A PDS5A SUGT1 MED29 PSMF1 PCGF1 UBE2H F RBM38 OTUB1 ADCY7 UBE3D BEND3 EIF4A1 TPRKB SUSD6 EIF4E2 HDAC3 CNOT4 EXOC2 CNOT8 KCMF1 CNOT2 SBNO2 EIF4G1 PIK3C3 CMTR1 AAGAB GANAB NCOR1 MPDU1 WDR48 CHMP3 CARM1 IFNAR2 NUFIP2 HIVEP1 PMPCB A ZNF737 ZNF654 ZNF296 NFKBIA MPLKIP RPS27L PICALM FBXO TSEN34 DYNLL1 VPS33A VPS26A PA PA TM9SF3 PPP3R1 PPP1R8 C2CD2L F UBE2E1 AM208B UBE2D3 UBE2D2 DNAJC3 POU2F1 RAD23B B3GNT2 N6AMT1 UBE2G2 UBE2G1 TGFBR1 TXNRD1 NDUFB4 ADAM10 ARPC1A IRF2BP2 ZCCHC7 AMBRA1 C10orf76 CREBBP C16orf62 ZFP36L1 HNRNPF CORO1A TSC22D2 F PRPF38A KIAA0922 CTDSPL2 G G TMEM160 TMEM127 RABGEF1 HNRN KHDRBS1 ARHGEF2 RALGAPB PYROXD1 HNRNPH1 SMARCD1 EIF4ENIF1 HSD17B10 ANKRD33B

TMD8 2.5 g

2.0

1.5

1.0

0.5 fraction MHC-I remainin

0.0 1 1 T2 A1 ID2 FLII GPI P AF8 ZFX AP1 HTT RFK TFG EED B2M IRF8 IRF2 HGS SPI1 SPIB TA T F8A1 PPIG JAK1 T UFL1 AKT1 LCP1 TYK2 PFN1 RFX5 EZH2 TSC2 CD70 BAP1 TSC1 TCH1 TCH8 OAZ1 USP1 UBA5 PDCL RCE1 LIPT1 CALR BRD9 CYLD EIF3L GPS2 APBP EDC4 RER1 ACTB XPO6 NSD1 XPOT PREP SOD1 GRB2 BRAP SPOP ANCB KDM8 EIF3K YRM5 TYMS SUOX CHUK SMG7 BBIP1 BCOR SIAH2 HIPK1 RABIF HLA-B DOHH S CHIC2 TP5F1 EP300 MOGS TTC14 LRP10 ZBTB2 AP2A1 TXNL1 SPPL3 PEX13 TRAF3 RAB14 RAB10 LSM12 NAA25 CSTF2 PTBP1 RAB35 T CTBP1 KEAP1 STUB1 SRSF6 CDK13 DDX23 L RBBP7 NLRC5 SURF4 AM98B LNPEP AP1M1 ERAP1 RAB7A UBE3A PDS5A TPRKB SUSD6 SUGT1 BEND3 ADCY7 PSMF1 F MED29 PCGF1 UBE3D UBE2H OTUB1 EIF4A1 RBM38 EIF4E2 KCMF1 CNOT4 CNOT8 CNOT2 SBNO2 EXOC2 HDAC3 CMTR1 EIF4G1 PIK3C3 AAGAB MPDU1 GANAB NCOR1 WDR48 CARM1 CHMP3 NUFIP2 IFNAR2 PMPCB HIVEP1 A ZNF296 ZNF737 ZNF654 NFKBIA MPLKIP PICALM RPS27L FBXO TSEN34 DYNLL1 VPS33A VPS26A PA PA C2CD2L F PPP1R8 UBE2E1 PPP3R1 TM9SF3 AM208B DNAJC3 UBE2D2 POU2F1 UBE2D3 RAD23B B3GNT2 N6AMT1 TGFBR1 UBE2G1 UBE2G2 TXNRD1 NDUFB4 ADAM10 ARPC1A ZCCHC7 IRF2BP2 AMBRA1 ZFP36L1 C16orf62 C10orf76 CREBBP HNRNPF CORO1A TSC22D2 F PRPF38A CTDSPL2 KIAA0922 G G TMEM127 TMEM160 RABGEF1 HNRN ARHGEF2 KHDRBS1 RALGAPB PYROXD1 HNRNPH1 SMARCD1 EIF4ENIF1 HSD17B10 ANKRD33B

3.0 BJAB

g 2.5

2.0

1.5

1.0

0.5 fraction MHC-I remainin

0.0 1 1 T2 A1 ID2 GPI FLII P AF8 AP1 ZFX HTT RFK TFG B2M EED IRF8 IRF2 HGS SPI1 SPIB TA T JAK1 PPIG F8A1 T UFL1 AKT1 TYK2 LCP1 RFX5 TSC1 TSC2 CD70 PFN1 EZH2 BAP1 TCH8 TCH1 USP1 OAZ1 UBA5 CALR NSD1 LIPT1 BRD9 EIF3L ACTB APBP PDCL RCE1 EDC4 CYLD XPO6 RER1 GPS2 BRAP PREP SOD1 GRB2 XPOT KDM8 SPOP YRM5 ANCB EIF3K TYMS CHUK SUOX BBIP1 SMG7 BCOR SIAH2 HIPK1 RABIF HLA-B DOHH S TP5F1 CHIC2 EP300 MOGS TTC14 LRP10 TXNL1 PEX13 ZBTB2 SPPL3 AP2A1 RAB10 PTBP1 CSTF2 LSM12 NAA25 RAB14 RAB35 TRAF3 T SRSF6 DDX23 CTBP1 KEAP1 CDK13 STUB1 L NLRC5 ERAP1 SURF4 RBBP7 AM98B LNPEP UBE3A RAB7A AP1M1 PDS5A SUGT1 TPRKB ADCY7 EIF4A1 PSMF1 RBM38 MED29 BEND3 PCGF1 UBE3D F OTUB1 UBE2H SUSD6 EIF4E2 KCMF1 CNOT4 HDAC3 CNOT2 CNOT8 EXOC2 SBNO2 CMTR1 EIF4G1 PIK3C3 AAGAB GANAB NCOR1 WDR48 MPDU1 CHMP3 CARM1 PMPCB IFNAR2 NUFIP2 HIVEP1 A ZNF296 ZNF737 ZNF654 NFKBIA MPLKIP PICALM RPS27L FBXO DYNLL1 TSEN34 VPS33A VPS26A PA PA PPP3R1 F C2CD2L TM9SF3 PPP1R8 UBE2E1 AM208B DNAJC3 RAD23B UBE2D3 POU2F1 UBE2D2 B3GNT2 N6AMT1 UBE2G1 UBE2G2 TGFBR1 TXNRD1 NDUFB4 ADAM10 ARPC1A ZCCHC7 IRF2BP2 CREBBP AMBRA1 C10orf76 ZFP36L1 C16orf62 HNRNPF CORO1A TSC22D2 F PRPF38A CTDSPL2 KIAA0922 G G TMEM160 TMEM127 RABGEF1 HNRN KHDRBS1 ARHGEF2 RALGAPB PYROXD1 HNRNPH1 SMARCD1 EIF4ENIF1 HSD17B10 ANKRD33B

2.5 SUDHL5 g 2.0

1.5

1.0

0.5 fraction MHC-I remainin

0.0 1 1 T2 A1 ID2 GPI FLII P AF8 AP1 ZFX HTT TFG RFK EED B2M HGS IRF2 IRF8 SPI1 SPIB TA T F8A1 PPIG JAK1 T UFL1 LCP1 TYK2 AKT1 TSC1 TSC2 PFN1 EZH2 RFX5 CD70 BAP1 TCH8 TCH1 OAZ1 UBA5 USP1 APBP ACTB EDC4 CALR NSD1 RCE1 CYLD EIF3L GPS2 LIPT1 RER1 XPO6 BRD9 PDCL PREP XPOT GRB2 SOD1 BRAP TYMS ANCB YRM5 SPOP EIF3K KDM8 SMG7 CHUK SUOX BBIP1 BCOR HIPK1 SIAH2 DOHH HLA-B RABIF S CHIC2 TP5F1 EP300 MOGS LRP10 TTC14 ZBTB2 AP2A1 TXNL1 SPPL3 PEX13 CSTF2 TRAF3 PTBP1 RAB14 RAB10 RAB35 LSM12 NAA25 T STUB1 CTBP1 CDK13 SRSF6 KEAP1 DDX23 L AP1M1 LNPEP NLRC5 SURF4 ERAP1 RBBP7 AM98B RAB7A PDS5A UBE3A OTUB1 PCGF1 SUSD6 MED29 TPRKB PSMF1 RBM38 F BEND3 UBE2H EIF4E2 SUGT1 ADCY7 UBE3D EIF4A1 CNOT2 KCMF1 CNOT8 SBNO2 CNOT4 HDAC3 EXOC2 AAGAB PIK3C3 EIF4G1 CMTR1 CARM1 CHMP3 GANAB NCOR1 WDR48 MPDU1 HIVEP1 IFNAR2 PMPCB NUFIP2 A ZNF296 ZNF737 ZNF654 NFKBIA MPLKIP PICALM RPS27L FBXO DYNLL1 TSEN34 VPS33A VPS26A PA PA TM9SF3 AM208B UBE2E1 F C2CD2L PPP3R1 PPP1R8 UBE2D3 DNAJC3 B3GNT2 RAD23B POU2F1 UBE2D2 TGFBR1 N6AMT1 UBE2G1 UBE2G2 NDUFB4 ADAM10 TXNRD1 ARPC1A ZCCHC7 IRF2BP2 AMBRA1 CREBBP C10orf76 ZFP36L1 C16orf62 HNRNPF CORO1A TSC22D2 F PRPF38A CTDSPL2 KIAA0922 G G TMEM127 TMEM160 RABGEF1 HNRN RALGAPB ARHGEF2 KHDRBS1 PYROXD1 HNRNPH1 SMARCD1 EIF4ENIF1 HSD17B10 ANKRD33B gene KO Supplemental Figure 3. Summary of cell specific effects of gene KOs on MHC-I. The fold change in surface MHC class I with KO of the indicated 199 genes, displayed across each cell model used for validation studies. See also Supplemental Table 3. TMD8 HLY1

sgRNA: AAVS1 AAVS1 SUGT1 CIITA blot: sgRNA: AAVS1 AAVS1 SUGT1 CIITA blot:

SUGT1 SUGT1

whole cell GAPDH GAPDH lysates

MHC-II MHC-II

CIITA CIITA CIITA IP/Western

CIITA Ab: - CIITA Ab: - + + + + + +

Supplemental Figure 4. SUGT1 controls steady state levels of MHC-II. TMD8 and HLY1 cells were infected with sgRNA against AAVS1 (negative control), SUGT1, or CIITA, and lysates were prepared for Western blotting. For detection of CIITA, IP/Western was required – independent antibodies were used for IP and blotting.

Supplemental Figure 5. Extension of data related to Figure 5. (A) Cell surface area was calculated by diameter measurements made by an automated cell counter and plotted against relative surface MHC- I levels (normalized to WT HBL1 cells). GCBs orange; ABCs blue. (B) Representative staining of IFNg receptor is shown for HBL1 and TMD8 relative to isotype control. (C) A panel of 7 DLBCLs all show significant IFNg receptor expression over isotype control staining, regardless of ABC/GCB classification. (D) The indicated cells were treated with 500U/mL of recombinant IFNg for 4 minutes, 15 minutes, or left untreated prior to cell lysate preparation. Western blotting was performed with the indicated antibodies. (E) HLA-B gene and promoter region from published IRF4 ChIP-seq datasets of lymphoblastoid GM12878 ( Omnibus, GSM803390). 18 18 19 HLA-B 19 HLA-B 19 HLA-B HLA-C 19 HLA-C HLA-C

17 17 18 18 18 18

Gene expression vs CNV

17 17 17 16 17 16 574 DLBCL patient cohort

16 16 16 15 16 15 ABC tumors GCB tumors

15 15 15 14 15 14 unclassified tumors Gene Expression

14 14 14 13 14 13 <0.0001 0.0123 0.0726 <0.0001 0.0875 0.2031

13 13 13 12 13 12 0 2 4 6 0 1 2 3 4 5 0 1 2 3 4 5 0 2 4 6 0 1 2 3 4 5 0 1 2 3 4 5

13 18 18 16 22 B2M 22 B2M 22 B2M 13 EZH2 EZH2 13 EZH2 IRF8 IRF8 IRF8

12 12 12 16 16

20 20 20 14

11 11 11

14 14

18 18 18 10 10 10 12

12 12

9 9 9

16 16 16 10 Gene Expression 10 10 8 8 8 <0.0001 0.1495 0.0409 0.0005 <0.0001 0.0463 <0.0001 0.0003 0.5075

14 14 14 7 7 7 8 8 8 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5 0 2 4 6 0 2 4 6 0 2 4 6 0 1 2 3 4 5 0 2 4 6 8 10 0 2 4 6

13 13 13 16 16 16 RFXAP 12 RFXAP 12 12 RFXAP CTDSPL2 CTDSPL2 CTDSPL2 PDIA3 PDIA3 PDIA3

12 12 12

15 15 15

10 11 11 11 10 10

10 10 10 14 14 14

9 9 9 8 8 8

13 13 13 8 8 8

6 6 6 7 7 7 Gene Expression 12 12 12

6 6 6 <0.0001 <0.0001 0.0054 <0.0001 0.1004 0.1756 <0.0001 0.0002 <0.0001

4 4 4 5 5 5 11 11 11 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 0 1 2 3 4 5 0 1 2 3 4 0 2 4 6 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5

16 B3GNT2 16 B3GNT2 16 B3GNT2 16 TAP1 16 TAP1 16 TAP1 19 HLA-A 19 HLA-A 19 HLA-A

18 18 18 14 14 14

14 14 14

17 17 17

12 12 12

12 12 12 16 16 16

10 10 10

15 15 15

10 10 10 Gene Expression 8 8 8 14 14 14 0.0112 <0.0001 0.0003 <0.0001 0.0002 0.0079 <0.0001 0.2055 0.4503

6 6 6 8 8 8 13 13 13 0 2 4 6 0 5 10 15 0 5 10 15 20 0 2 4 6 0 1 2 3 4 5 0 1 2 3 4 5 0 2 4 6 0 1 2 3 4 5 0 1 2 3 4 5 3060

15 15 15 12 12 12 CD70 CD70 CD70 ZNF296 ZNF296 ZNF296 15 RFXANK 16 RFXANK 16 RFXANK

10 10 10 14 14

10 10 10 10

8 8 8 12 12

5 5 5 5

6 6 6 10 10 Gene Expression

<0.0001 <0.0001 0.0003 <0.0001 0.0231 0.0810 <0.0001 0.0083 0.0027

0 0 0 4 4 4 0 8 8 0 1 2 3 4 0 1 2 3 4 0 5 10 15 0 2 4 6 0 2 4 6 0 2 4 6 0 1 2 3 4 0 1 2 3 4 5 0 2 4 6

15 TAP2 TAP2 15 TAP2 14 AP2A1 14 AP2A1 14 AP2A1 16 NLRC5 16 NLRC5 16 NLRC5

14 13 14 14 14

13 13 12 12 12

12 12 12

12 11 Gene Expression 10 Gene Expression 10 Gene Expression 10

11 11 10 10 10 Gene Expression 8 8 8 10 9 <0.0001 <0.0001 <0.0001 <0.0001 0.1384 0.0855 <0.0001 0.0317 0.5963

9 9 8 8 8 6 6 6 0 2 4 6 0 1 2 3 4 5 0 1 2 3 4 5 0 2 4 6 8 0 2 4 6 0 2 4 6 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 gene copy number Supplemental Figure 6. Copy number variation correlation with gene expression in DLBCL patient tumors. Gene copy number variation is plotted versus gene expression for DLBCL patient tumor samples, indicating significant mRNA changes upon genetic copy number changes. p-values represent likelihood of positive correlation. A

HLA-A HLA-E TUBB DDR1 CDSN HLA-B LTA MSH5 C2 FKBPL BTNL2 HLA-DQB1 TAP2 COL11A2 DHX16 VARS2 CCHCR1 MICA TNF HSPA1L CFB AGER HLA-DRA PSMB8 RGL2 C6orf15 MICB DDAH2 SKIV2L NOTCH4 HLA-DRB5 TAP1 TAPBP PSORS1C1 DDX39B HSPA1A DXO HLA-DRB1 PSMB9 ZBTB22 POU5F1 NFKBIL1 HSPA1B C4A HLA-DQA1 HLA-DMB HLA-C NCR3 NEU1 C4B HLA-DOA PRRC2A EHMT2 HLA-DPB1 BAG6 CYP21A2 APOM TNXB GPANK1 SLC44A4 1 Mb hg19 30,500,000 31,000,000 31,500,000 32,000,000 32,500,000 33,000,000

chr6 (p22.1-p21.32) 6p22.3 6q12 q13 q14.1 q15 16.1 6q21 266q27 Scale 50 Mb hg19 chr6: 50,000,000 100,000,000 150,000,000

B PDIA3 WDR76 CASC4 EIF3J PATL2 NCBI RefSeq genes ELL3 FRMD5 CTDSPL2 PATL2 SERF2 SPG11 B2M SERINC4 HYPK MFAP1

chr15 (q15.3-q21.1) p13 15p12 15p11.2 q11.2 12 15q14 21.1 q21.3 22.2 15q23 25.3 q26.1 26.2 26.3

Scale 50 Mb hg19 chr15: 10,000,000 50,000,000 100,000,000

C

0.8 MCD N1 A53 Composite

n BN2 ST2 EZB Other

0.6 Alteratio

0.4

0.2 Fraction of Cases with

0.0 Amp Amp Amp Amp

Mut HL Mut HL Mut HD HL Mut HD HL Mut HD HL Mut HD HL Mut HD HL Mut HD HL Mut HD HL Mut HD HL Mut HD HL Mut HD HL Mut HD HL Mut HD HL Mut HD HL Mut Gain Mut Gain Mut Gain Mut Gain B2M IRF8 TAP1 TAP2 RFX5 CD70 EZH2 PDIA3 HLA-B HLA-A HLA-C AP2A1 TAPBP NLRC5 RFXAP ZNF296 B3GNT2 RFXANK CTDSPL2 NCBI RefSeq genes

Supplemental Figure 7. Patient chromosomal deletions and subtype classifications of mutations. (A) Summary of chromosomal losses in patient tumors at the MHC , showing obvious selection of losses in APP machinery. (B) Same as A, but with 15. (C) Frequency of mutation in indicated genes across DLBCL patient cohort, sorted by LymphGen subclassification. Mut, mutation; HD, homozygous deletion; HL, heterozygous loss; gain, single copy gain; amp, multiple copy gain. A Fold change MHC-I (SUDHL4) Fold change MHC-I (DB)

5

5 4 4

1 3 1 3

2 2 2 2 3 3 4 5 4 5 1 1

6 0 6 0 compound titration compound titration compound titration compound titration compound titration compound titration

B target: EZH2

H 1 2 N O CH3 O H Cl O O O N

N NH N CH O N 3 O N N H3C CH3 Cl H3C N

CH3 PF-06821497 Pfizer, phase I trials SCLC, castration resistant prostate UNC1999 , follicular lymphoma preclinical

target: TYMS 3 4 5 6

Pemetrexed (Alimta) Trifluridine (Lonsurf) Floxuridine (FUDR) Raltitrexed (Tomudex) FDA approved in 2004 FDA approved in 2015 FDA approved in 1970 Approved in 1998 Non-squamous non-SCLC Colorectal cancer Kidney cancer in Canada and EU Pleural mesothelioma in combination with tipiracil Stomach cancer Colorectal cancer

Supplemental Figure 8. Targeted drug screen for inhibitors of negative MHC-I regulators. (A) 48 selected small molecules were dry-spotted into wells of 384-well plates in a 7-dose titration series as indicated, spanning 5.7nM – 23.5 µM final concentrations upon cell plating. Cells were grown for 2 days prior to flow cytometry analysis of MHC-I surface expression. Fold change in surface MHC-I relative to vehicle controls is plotted by heatmap. Compounds 1-6, highlighted in black boxes, showed consistent, dose-dependent upregulation of MHC-I in both cell lines tested. (B) Chemical structures and clinical information related to compounds 1-6 above.

A B donor 1 anti-NY-ESO-1 T-cells donor 2 anti-NY-ESO-1 T-cells RL DB 9000 1500 50 +SUDHL4 EV 80 +SUDHL4 EV

n +SUDHL4 NY-ESO-1 +SUDHL4 NY-ESO-1 n Z Z 40 A A 60 T 6000 T 1000 30 40 20 3000 500 -cell activatio 2 -cell activatio T R = 0.9761 T class I MFI, 2 class I MFI, 20 R = 0.8697 10 % %

0 0 0 0 0 3000 6000 9000 0 500 1000 1500 1:81 1:27 1:9 1:3 1:1 1:81 1:27 1:9 1:3 1:1 class I MFI, GSK126 class I MFI, GSK126 target:effector ratio target:effector ratio

C expected WT sequence expected WT sequence

expected coding sequence expected coding sequence

Karpas422 BJAB

A/T SUDHL4 SUDHL5

G/T WSU-DLCL2 HBL1

A/T D EZH2 mutant: EZH2 WT (Y641):

OCI-LY18 TMD8 Vallois NUDHL1 Karpas422 (het: Y641 / N641) SUDHL5 U2932 HS602 SUDHL4 (het: Y641 / S641) OCI-LY19 OCILY3 PR1 WSU-DLCL2 (het: Y641 / F641) Daudi BJAB RIVA SR786 OCI-LY1 (het: Y641 / N641) MD901 OCI-LY8 HBL1 TOLEDO CARNAVAL (het: Y641 / F641) Namalwa SUDHL7 HLY1 Will1 DB (het: Y641 / N641) OZ SUDHL2 PCNS-TK RL (het: Y641 / N641) SLVL SK1 OCI-LY10 SUDHL10 (het: Y641 / F641) DLBCL1 DLBCL2 SUDHL6 (het: Y641 / N641) MWCL-1 WSU-NHL Jeko RPC1-WM1 WSU-FSCCL UPN1 WSU-WM SC1 OYB (het: Y641 / C641) SUDHL16 Dogkit DOHH2 Y641, but evidence of other mutations: NUDUL1 Pfeiffer (A677 / G677) MC116 HF (A687 / G687)

Supplemental Figure 9. Extension of EZH2-related data in Figure 7. (A) Treatment of DB or RL cells with increasing doses of tazemetostat or GSK126 for 7 days (DMSO, 0.4, 1.5, and 4µM). (B) Confirmation of NY-ESO-1 specificity in transduced SUDHL4 lines. T cells were stained for 4-1BB upregulation after co-culture with SUDHL4 cells transduced with either empty vector (EV) or NY-ESO-1 at the indicated target:effector ratios. (C) Example chromatograms of the EZH2 gDNA near Y641. Homozygous Y641 lines and heterozygous Y641 & Y641N/S/F are clearly distinguished. (D) Summary of EZH2 mutational status at Y641 across a number of tumor lines, with indicated heterozygous mutations in red. A B Carnaval DB SUDHL4 nM pemetrexed: 9 327688192 2048 512 128 32 8 2 0 7 100 4 +H O 8 +H O +H O 2 7 2 2 SUDHL4 +PEM +PEM 6 +PEM 75 DB e 3 e e 5 2 SUDHL5 50 4 2 3 WSU-DLCL2 25

fold chang fold chang 1 fold chang 2 Carnaval 1 0 1 HBL1 % live cells relative to 0 0 0 TMD8 vehicle

CD86 CD20 CD45 live % MHC-I MHC-II MHC-I CD86 CD20 CD45 live % MHC-I CD86 CD20 MHC-II CD45 live % surface area surface area surface area

Supplemental Figure 10. Extension of TS-related data in Figure 7. (A) The indicated DLBCLs were cultured with serial dilutions of the TS inhibitor pemetrexed to determine % growth inhibition after 48 hours. (B) Cells were treated with pemetrexed or vehicle control and stained for various surface markers after 48 hours (Carnaval, 500nM; DB, 100nM; SUDHL4, 500nM). Surface areas were calculated by automated diameter measurements; “% live” indicated relative fraction of cells live by FACS scatter. For entire figure, bar graphs represent mean with standard deviations, minimum n = 3.