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1 doi:1.138/nture17 Men tumour dimeter (mm) 2 Rg2-/ Control IgG!-CD8!-CD c Men tumour dimeter (mm) d Ifnr1-/- Rg2-/ Ifngr1-/- d42m1!ic Dys post trnsplnt Supplementry Figure 1. d42m1 is highly immunogenic unedited srcom. d42m1 tumour cells were injected sucutneously t dose of 1x1 6 cells into syngeneic wild type (-d), Rg2 -/- ( nd c), Ifnr1 -/- (c), or Ifngr1 -/- (d) mice nd tumour growth ws followed over time., Groups of WT mice injected with 1x1 6 d42m1 tumour cells were treted with control IgG, nti- CD4, or nti-cd8α mas (2 µg) strting t dy -1 nd then every 7 dys therefter. d, d42m1 tumour cells were rendered insensitive to IFN-γ (d42m1δic) y over-expressing dominnt-negtive version of IFNGR1 (IFNGR1ΔIC) nd were then trnsplnted (1x1 6 cells) into WT mice. Dt re presented s verge tumour dimeter ± s.e.m. of 3- mice per group nd re representtive of t lest three independent experiments. Smples were compred using n unpired, two-tiled Studentʼs t test (p<., p<.1, nd p<.1).! 1

2 RESEARCH Men tumour dimeter (mm) Rg2-/ Control IgG!-CD8!-CD4 c Men tumour dimeter (mm) d 2 Ifnr1-/- 2 Rg2-/- 1 Ifngr1-/- H31m1!IC Dys post trnsplnt Supplementry Figure 2. H31m1 is highly immunogenic unedited srcom. H31m1 tumour cells were injected sucutneously t dose of 1x1 6 cells into syngeneic wild type (-d), Rg2 -/- ( nd c), Ifnr1 -/- (c), or Ifngr1 -/- (d) mice nd tumour growth ws followed over time., Groups of WT mice injected with 1x1 6 H31m1 tumour cells were treted with control IgG, nti-cd4, or nti-cd8α mas (2 µg) strting t dy -1 nd then every 7 dys therefter. d, H31m1 tumour cells were rendered insensitive to IFN-γ (H31m1ΔIC) y over-expressing dominnt-negtive version of IFNGR1 (IFNGR1ΔIC) nd were then trnsplnted (1x1 6 cells) into WT mice. Dt re presented s verge tumour dimeter ± s.e.m. of 3- mice per group nd re representtive of t lest three independent experiments. Smples were compred using n unpired, two-tiled Studentʼs t test (p<., p<.1, nd p<.1).! 2

displyed for the MCA srcom cell lines nd norml skin firolsts tht

3 RESEARCH Supplementry Figure 3. Exome sequencing coverge. Percentge of exome sequence coverge (2x, x, 1x, x, 1x, x) is displyed for the MCA srcom cell lines nd norml skin firolsts tht were isolted from three independent syngeneic 129/Sv Rg2 -/- mice.! 3

4 RESEARCH Anti-d42m1 CTL Line" Anti-H31m1 CTL Line" 3 4 IFN-γ (pg/ml)" H31m1 d42m1 F H31m1 d42m1 F % Specific lysis" E:T rtio" Supplementry Figure 4. H31m1 nd d42m1 scroms re immunogeniclly distinct. IFN-γ ELISA ssy () nd killing ssy () using the ulk CTL lines developed ginst either prentl d42m1 (left pnel) or H31m1 (right pnel) nd tested ginst unedited MCA srcom cell lines, d42m1, H31m1, F1, 1773, 1782, nd 1779 or the edited F244 MCA srcom derived from wild type 129/Sv mouse. Dt re representtive of t lest two independent experiments nd re presented s verge IFN-γ relese () or percent of tumour cell lysis () ± s.e.m. Smples were compred using n unpired, two-tiled Studentʼs t test (p<., p<.1, nd p<.1)." 4

5 RESEARCH Men tumour dimeter (mm) Rg2-/ d42m1-rgpss d42m1-es Dys post trnsplnt c 1 1/1" d 1 9/9" 1/1" 1/1" Tumour Growth (%) Tumour Growth (%) 8 6 4/22" 4 2/1" 2 Rg2-/- d42m1-rgpss d42m1-es1 d42m1-es2 d42m1-es3 Supplementry Figure. Escpe tumours of d42m1 disply reduced immunogenicity nd n edited growth phenotype., Growth of d42m1 tumour cells (1x1 6 ) following trnsplnttion into wild type (solid lines) or Rg2 -/- (dshed lines) mice., Growth of the escpe tumour d42m1-es3 (hrvested from the wild type mouse ering n escped d42m1 tumour in nd generted into cell line) (n=, dimonds) or d42m1-rgpss (n=, squres) following trnsplnttion (1x1 6 cells) into WT mice. Dt presented s verge tumour dimeter ± s.e.m of mice per group over time. c, Summry of tumour growth in WT mice or Rg2 -/- mice were chllenged with 1x1 6 d42m1 tumour cells. Dt presented s percent tumour positive from 2-4 independent experiments (n=4-6 mice per group). d, Summry of tumour growth in WT mice chllenged with 1x1 6 d42m1-rgpss, d42m1- es1, d42m1-es2, or d42m1-es3 tumour cells. Dt presented s percent tumour positive from two independent experiments (n=4- mice per group). Smples were compred using n unpired, two-tiled Studentʼs t test (p<.1 nd p<.1)."

6 RESEARCH 2 d42m1-t4" d42m1-t" d42m1-t6" Men tumour dimeter (mm) d42m1-t7" d42m1-t8" Dys post trnsplnt Supplementry Figure 6. Growth phenotypes of dditionl regressor d42m1 clones. Tumour growth in WT mice chllenged with 1x1 6 d42m1-t4, d42m1-t, d42m1-t6, d42m1-t7, or d42m1-t8 tumour cells. Dt presented s verge tumour dimeter ± s.e.m of mice per group over time nd re representtive of t lest two independent experiments." 6

cells, d42m1 regressor clones (red), d42m1

(lue) is displyed in n unrooted phylogenetic tree

7 RESEARCH Supplementry Figure 7. d42m1 tumour cell clones nd escpe tumours re genomiclly relted., Numer nd type of somtic, non-synonymous muttions in d42m1 relted tumour cells., Reltedness of norml firolsts, H31m1, d42m1 prentl cells, d42m1 regressor clones (red), d42m1 progressor clones (green) nd d42m1 escpe tumours (lue) is displyed in n unrooted phylogenetic tree generted from exome sequencing dt using PHYLogeny Inference Pckge (PHYLIP). Numers represent the degree of reltedness etween smples" 7

RESEARCH c WT 4 p Spectrin-β2 Mut 2 p Supplementry Figure 8.

, Numer of WT or mutnt spectrin-β2 reds from Illumin cdna CpSeq., cdnas from prentl d42m1 or d42m1 tumour cell clones were prepred.

The G to T point muttion in spectrin-β2 cuses novel restriction site, such tht only the R913L muttion will e cleved y Pst1 restriction enzymes.

8 RESEARCH c WT 4 p Spectrin-β2 Mut 2 p Supplementry Figure 8. The R913L spectrin-β2 muttion in d42m1 tumour cells is tumour-specific muttion nd not host polymorphism., Numer of WT or mutnt spectrin-β2 reds from Illumin cdna CpSeq., cdnas from prentl d42m1 or d42m1 tumour cell clones were prepred. After RT-PCR mplifiction, cdna frgments were cut y restriction enzyme Pst1 nd nlyzed y electrophoresis in 1.2% grose gels. The G to T point muttion in spectrin-β2 cuses novel restriction site, such tht only the R913L muttion will e cleved y Pst1 restriction enzymes. c, A frozen d42m1 tumour iopsy from the originl tumour ws thwed nd sorted into CD4 - cells (primry d42m1 tumour cells) nd CD4 + cells (tumour-infiltrting leukocytes (TILs)). RNA ws isolted from sorted cells for cdna synthesis, which ws mplified using primer pirs for spectrin-β2 nd digested y Pst1 s in (). The prentl d42m1 cell line nd the norml skin firolst line SK1. which ws derived from syngeneic 129/Sv Rg2 -/- mouse were lso included s positive nd negtive controls, respectively. Also, these sorted smples were sequenced using 373 nd the results re presented in Supplementry Tle 4." 8

9 Men tumour dimeter (mm)" Men tumour dimeter (mm)" RESEARCH Reltive expression of " Mutnt spectrin-β2" 2 1 " " " " " " d42m1-es3-gfp/ spectrin-!2 WT.1 d42m1-es3-gfp/ spectrin-!2 mu Dys post trnsplnt" c Tumour growth (%)" /1 1/1 1/1 d42m1-es3 GFP/spectrin-β2 / 2/ d d42m1-es3-gfp/spectrin-β2 mu.14" Rg2-/ Dys post trnsplnt" d42m1-es3 GFP/spectrin-β2 Supplementry Figure 9. Enforced expression of mutnt spectrin-β2 in d42m1 escpe tumour cells results in rejection in wild type mice., qrt- PCR nlysis using primer pir specific for mutnt spectrin-β2 in d42m1-es3 tumour cell clones tht hve een engineered to express either wild type or mutnt spectrin-β2. Dt is displyed s reltive expression fter normliztion to control GAPDH expression nd is representtive of three independent experiments., d, Growth of d42m1-es3 tumour cell clones reconstituted with WT or mutnt spectrin-β2 nd control d42m1-es3 expressing only GFP following trnsplnttion of 1x1 6 cells into five-memer groups of WT (, d) or Rg2 -/- mice (d). Dt re presented s verge tumour dimeter ± s.e.m. over time. c, Percent tumour positive mice in five-memer groups of WT mice from 2-3 independent experiments is shown. Smples were compred using n unpired, two-tiled Studentʼs t test (p<., p<.1, nd p<.1; n.s. is non-significnt)." 9

10 RESEARCH Reltive expression of " Mutnt spectrin-β2" " d42m1-t9 d42m1-t3 d42m1-t1 d42m1-t9 d42m1-t3 d42m1-t1 d42m1 d42m1-es1 d42m1-es2 d42m1-es3 d42m1-es1 d42m1-es2 d42m1-es3 TSA+Az TSA+Az Supplementry Figure 1. Lck of mutnt spectrin-β2 mrna in d42m1 tumour clones or escpe tumours is not due to epigenetic silencing. d42m1 tumour cell clones (left) nd d42m1 escpe tumours (right) were treted with Trichosttin A (TSA) (.3 µmol/l) nd -zcytidine (Az) ( µmol/l) for 3 dys prior to cdna preprtion. Rel-time PCR using specific primer pir for mutnt spectrin-β2 ws performed. Dt is displyed s reltive expression fter normliztion of control GAPDH expression nd is representtive of 3 independent experiments. Smples were compred using n unpired, two-tiled Studentʼs t test (p<.1; n.s. is non-significnt)." 1

RESEARCH H-2D (19) Affinity Vlue [(1/IC) x 1] H-2K (8) Mutnt Epitopes c 1/1 1/1 IFN-γ (pg/ml)" Tumour growth (%) 4/22

, Missense muttions for H31m1 were nlyzed for potentil MHC clss I neoepitopes tht ind to either H-2D or H-2K.

Numer of potentil strong ffinity inders for ech MHC clss I molecule re indicted in prentheses.

11 RESEARCH H-2D (19) Affinity Vlue [(1/IC) x 1] H-2K (8) Mutnt Epitopes c 1/1 1/1 IFN-γ (pg/ml)" Tumour growth (%) 4/22 /4 d42m1 H31m1 Supplementry Figure 11. H31m1 tumour cells express numer of potentil ntigens for H-2D nd H-2K., Missense muttions for H31m1 were nlyzed for potentil MHC clss I neoepitopes tht ind to either H-2D or H-2K. Predicted epitope inding ffinities were ultimtely expressed s Affinity Vlues (Affinity Vlue = 1/IC X 1). Numer of potentil strong ffinity inders for ech MHC clss I molecule re indicted in prentheses., IFN-γ production y n H31m1-specific CD8 + CTL line following incution with H31m1 trget cells with locking ntiodies ginst CD8, CD4, H-2D nd/or H-2K. c, Percent of tumour growth in Rg2 -/- or WT mice trnsplnted with d42m1 or H31m1 tumour cells (1 x 1 6 cells) from 2- independent experiments. Smples were compred using n unpired, two-tiled Studentʼs t test (p<., p<.1, nd p<.1)." 11

12 RESEARCH Supplementry Tle :! Predicted Strong Affinity Epitopes Specific for d42m1 Regressor Tumours! H-2D :! Peptides! IC (nm)! P! T1! T2! T9! Olfr19 (L14M)! YSVTNEFIL (wild-type)!! YSVTNEFIM (mutnt)!! Spn2 (R913L)! VAVVNQIAR (wild-type)! 3! VAVVNQIAL (mutnt)! 7! Tk1 (G722S)! GGLRNVDCL (wild-type)! 36! GSLRNVDCL (mutnt)! 8! Fm38 (M134I)! MAGINTDHL (wild-type)! 23! IAGINTDHL (mutnt)! 14! Cxx1 (D4N)! YMLVDDRTF (wild-type)! 433! YMLVNDRTF (mutnt)! 14! H2-Q6 (R142L)! FAYEGRDYI (wild-type)! 27! FAYEGLDYI (mutnt)! 14! A2m (G777V)! FCLSNDTGL (wild-type)! 2! FCLSNDTVL (mutnt)!! Uqln4 (R262L)! RALSNLESV (wild-type)! 1! LALSNLESV (mutnt)! 24! Olfr7 (D9N)! VSVVDTTFT (wild-type)! 119! VSVVNTTFT (mutnt)! 41! Dst (W449L)! AMMQWLEKM (wild-type)! 18! AMMQLLEKM (mutnt)! 4! Tcl1 (H146N)! AAPSHAAVA (wild-type)! 262! AAPSNAAVA (mutnt)! 48! H-2K :! Peptides! IC (nm)! P! T1! T2! T9! Gm77 (W61R)! VTFQYSWV (wild-type)! 6! VTFQYSRV (mutnt)! 2! Wscd1 (A183G)! RSYVYAGL (wild-type)! 2! RSYVYGGL (mutnt)! 3! Zdhhc22 (C137Y)! LACLYSMV (wild-type)! 683! LAYLYSMV (mutnt)!! Nckp1 (R917P)! VILSFRSL (wild-type)! 4! VILSFPSL (mutnt)!! 12

13 RESEARCH H-2K (continued):! Peptides! IC (nm)! P! T1! T2! T9! Olfr1394 (T284N)! VTPMFNPL (wild-type)! 6! VNPMFNPL (mutnt)!! Ep4.1l4 (H9L)! VGPAYALH (wild-type)! 3972! VGPAYALL (mutnt)! 6! Vipr2 (F298L)! IVVNFALF (wild-type)! 169! IVVNFALL (mutnt)! 7! Ac14 (A44G)! SAFLYGLV (wild-type)! 8! SGFLYGLV (mutnt)! 7! Oog3 (L22M)! VYFIYGNL (wild-type)! 6! VYFIYGNM (mutnt)! 7! Tmem194 (W24C)! RSYWHYLL (wild-type)! 9! RSYCHYLL (mutnt)! 8! Ts2r116 (K11N)! SIFYFFKI (wild-type)! 36! SIFYFFNI (mutnt)! 1! Cntnp3 (G382V)! SSGSYLAL (wild-type)! 97! SSVSYLAL (mutnt)! 11! Mettl4-ps1 (P3L)! SGFLYPLV (wild-type)! 7! SGFLYLLV (mutnt)! 13! Dnmt3 (R732P)! LFFEFYRL (wild-type)! 2! LFFEFYPL (mutnt)! 14! Pde2 (S42N)! ISIAHSLL (wild-type)! 8! INIAHSLL (mutnt)! 14! Got1l1 (L66M)! LDYEYLPL (wild-type)! 29! MDYEYLPL (mutnt)! 14! Cyp341 (V32I)! IVFIFAGY (wild-type)! 26! IIFIFAGY (mutnt)! 14! Gm1696 (E82Q)! SSTAYMEL (wild-type)! 37! SSTAYMQL (mutnt)!! Cyr2 (A68T)! VIRAYTPV (wild-type)! 28! VIRTYTPV (mutnt)! 18! Atg9 (C19F)! DTCSFAQM (wild-type)! 184! DTFSFAQM (mutnt)! 19! Nop6 (G194V)! YGYHFPEL (wild-type)! 22! YVYHFPEL (mutnt)! 2! Ogdh (V42I)! IVYETFHL (wild-type)! 3! IIYETFHL (mutnt)! 22! 13

14 RESEARCH H-2K (continued):! Peptides! IC (nm)! P! T1! T2! T9! Bco2M3I! MYYSMPFL (wild-type)! 11! IYYSMPFL (mutnt)! 24! Acd4V196L! SIFGYFIV (wild-type)! 7! SIFGYFIL! (mutnt)! 26! InppdM32L! VSFMFNGT (wild-type)! 23! VSFLFNGT (mutnt)! 28! Qsox2D64Y! FSSLDMSL (wild-type)! 868! FSSLYMSL (mutnt)! 28! Odz2V69F! VSFNTVVL (wild-type)! 84! VSFNTVFL! (mutnt)! 29! Adr1G3V! IVFWLGYL (wild-type)! 28! IVFWLVYL (mutnt)! 29! Ts2r113W26L! SLLIFSLW (wild-type)! 1743! SLLIFSLL! (mutnt)! 3! Prom1I61M! MVHIFLNV (wild-type)! 47! MVHMFLNV (mutnt)! 31! Rd9P281L! SSPQLLPL (wild-type)! 78! SSLQLLPL (mutnt)! 32! Psrc1R63L! VRFSLGPL (wild-type)! 27! VLFSLGPL (mutnt)! 33! StmpQ183P! IVQEFGKV (wild-type)! 9! IVPEFGKV (mutnt)! 34! Ryr2T168N! LQFHYHTL (wild-type)! 186! LQFHYHNL! (mutnt)! 34! Cmk2G21R! GVILYILL (wild-type)! 219! RVILYILL (mutnt)! 36! Thsd1G148V! GVFTTQPL (wild-type)! 283! VVFTTQPL (mutnt)! 37! Olfr114G96C! ISFVGCFV (wild-type)! 73! ISFVCCFV (mutnt)! 4! Trpc7G64V! VLYGVYNV (wild-type)! 186! VLYVVYNV (mutnt)! 41! Ccng8G79V! GGLTHSGL (wild-type)! 339! VGLTHSGL (mutnt)! 42! Grsf1S43C! ITMEYSSS (wild-type)! 3! ITMEYSSC! (mutnt)! 44! 14

15 RESEARCH H-2K (continued):! Peptides! IC (nm)! P! T1! T2! T9! Dnhc17 (M1234I)! IMRDLANL (wild-type)! 74! IIRDLANL (mutnt)! 47! Olfr122 (D181V)! INHFYCAD (wild-type)! 828! INHFYCAV (mutnt)! 49! Olfr1366 (L68F)! ANLSLVDL (wild-type)! 1667! ANLSFVDL (mutnt)!! Supplementl Tle. Predicted Strong Affinity Epitopes Specific for d42m1 Regressor Tumours. A list of ll epitopes tht re predicted to ind with strong ffinity (i.e. IC < nm), resulting from muttions present in ny regressor tumour smples, ut not in ny of the progressor tumour smples. The list order is sed on the predicted ffinity of the mutnt epitope to either H-2D or H-2K, eginning with the strongest predicted inder. Spn2 is the strongest H-2D epitope present in ll of the regressor smples. ʻPʼ = prentl d42m1; ʻT1 = d42m1-t1 clone; ʻT2ʼ = d42m1-t2 clone; ʻT9ʼ = d42m1-t9 clone; ʻGm77ʼ corresponds to Enseml gene product ENSMUSG73789; ʻGm1696ʼ corresponds to ENSMUSG7672.!

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION doi:1.138/nture1794 BR EPFs BRI1? ERECTA TMM BSKs YDA PP2A BSU1 BIN2 pbzr1/2 BZR1/2 MKK4/5/7/9 MPK3/6 SPCH Cell growth Stomtl production Supplementry Figure 1. The model of BR nd stomtl signling pthwys.