A 93‐VU‐147T cells

B HeLa cells

Supplementary Figure S1. Representative examples of short tandem repeat (STR) profiling. Panels A-B show a representative copy of the electropherogram and Cell Line DNA Typing report obtained following STR analysis by the accredited company NorthGene (Newcastle upon Tyne, UK). The authenticity was assessed by comparing the generated Short Tandem Repeat (STR) profile with the source STR profiles present in the American Type Culture Collection, Cellosaurus and the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). 3 (***, 43) = NC ttelrn ( larynx the at HNSCC NC ape e ugopaeshown. are subgroup per samples HNSCC ifrn usts ubro NC ape e ugopaeson S o infcn;***, significant; not NS, shown; are subgroup per samples HNSCC of Number subsites. different .0;Mn-hte test). Mann-Whitney 0.001; upeetr iueS2. Figure Supplementary U 5 NC tdfeetHSCaaoia usts(S o infcn;***, significant; not (NS, subsites shown. anatomical are HNSCC subgroup different per at HNSCC MUT A C D Distribution (%) PTTG Expression

P PTTG Expression 10 15 20 25 30 35 40 45 50 n 0 5 4.39x10 = 20 p53 WT 09 7284623 6 4 238 77 96 20 *** ayxOropharynx Larynx 96 NS n -18 MUT 1) rlcvt ( cavity oral 116), = p53 -,KuklWli test). Kruskal-Wallis K-W, ; E, C P itiuin()o P -e r(v)HSCa ifrn usts(***, subsites different at HNSCC (+ve) or (-ve) HPV of (%) Distribution TGepeso tHSCaaoia subsites. anatomical HNSCC at expression PTTG o hse lto TGepeso (log expression PTTG of plot whisker Box , 77 = 4.25x10 *** P 238 = 1.83x10

PTTG Expression -18 n (K-W) 4 -10 1) yohrn ( hypopharynx 315), = NS (K-W) rlcvt Hypopharynx Oral cavity p53 WT 6 MUT p53 WT p53 NS B, MUT p53 TGepeso tteoohrn versus oropharynx the at expression PTTG 56 *** 23 n 0 n rpayxsbie ( subsites oropharynx and 10) =

PTTG Expression B 2 numthdHSCwt Tp3o U 5 sidctdat indicated as p53 MUT or p53 WT with HNSCC unmatched in ) PTTG Expression p53 WT E A n o hse lto TGepeso (log expression PTTG of plot whisker Box , NS

P Distribution (%) 10 15 20 25 30 35 40 45 MUT .0;Fse’ xc et.Nme fHSCsamples HNSCC of Number test). exact Fisher’s 0.001; < p53 0 5 1 10 116 18 *** 3 315 P te usts(NS, subsites other P 0.001. < n PTTG Expression *** .0;Fse’ xc et.Nme of Number test). exact Fisher’s 0.001; < 9 oprdt omlseies( specimens normal to compared 79) = 45 *** *** p53 WT 2 56 D, *** NS itiuin()o Tp3or p53 WT of (%) Distribution MUT p53 79 8 o infcn;***, significant; not *** 32 HPV+ve HPV-ve 2 nunmatched in ) P < n PTTG PTTG-pT60

20x 80x 20x 80x

HNSCC Stage 1

HNSCC Stage 2

HNSCC Stage 3

HNSCC Stage 4

Normal

Supplementary Figure S3. Expression of PTTG in HNSCC. Representative images of total (left) and T60-phosphorylated (right) PTTG in HNSCC and normal tissue. Images taken at 20x (scale bars, 100 m) and 80x (scale bars, 10 m) magnification as indicated. Sections were counterstained for haematoxylin. Total and T60-phosphorylated PTTG protein were abundantly overexpressed in oropharyngeal tumours of all stages (1-4) as indicated. A B * NS Overall survival of HNSCC patients stratified on PBF expression

Patient N P P P stratification L B T

HIGH PBF (Q4) 129 vs 0.025 0.013 0.016 388 PBF Expression LOW PBF (Q1-Q3)

HIGH PBF (Q4) 129 vs NS 0.017 0.028 129 LOW PBF (Q1)

HIGH PBF (Q3Q4) 258 vs NS 0.030 NS 259 n 43116 315 10 79 LOW PBF (Q1Q2)

C D PBF PBF-pY174

20x 80x 20x 80x 28kDa PBF

44kDa -actin HNSCC Stage 1

HNSCC Stage 2

HNSCC Stage 3

HNSCC Stage 4

Normal

Supplementary Figure S4. Expression of PBF in HNSCC. A, Box whisker plot of PBF expression (log2) in unmatched HNSCC at the larynx (n = 116), oral cavity (n = 315), hypopharynx (n = 10) and oropharynx subsites (n = 79) compared to normal specimens (n = 43) (NS, not significant; *, P < 0.05; Kruskal-Wallis test). B, Overall survival for HNSCC TCGA patients based on stratification criteria of PBF expression as indicated. Numbers of patients per subgroup are shown as well as P-values determined using the indicated test (L=Log Rank, B-Breslow,

T=Tarone-Ware). Red box highlights a significant difference using the PB test in overall survival for all PBF stratification groups. In the PB test time points are weighted by the number of cases at risk at each time point which emphasizes shorter survival times. C, Western blot analysis of PBF expression in HNSCC cell lines used in study compared to HeLa cells. D, Representative images of total (left) and Y174- phosphorylated (right) PBF protein in HNSCC and normal tissue. Images taken at 20x (scale bars, 100 m) and 80x (scale bars, 10 m) magnification as indicated. Sections were counterstained for haematoxylin. Total and Y174-phosphorylated PBF protein were abundantly overexpressed in oropharyngeal tumours of all stages (1-4) as indicated. AB93-VU-147T 93-VU-147T

0 h 24 h 24 h 80 *** 80 ***

70 70

60 Scr 60 Scr shRNA shRNA 50 50

40 40

30 30

20 20 % Wound Recovery Wound % Recovery Wound % 10 10 PBF PTTG shRNA 0 shRNA 0

shRNA shRNA

C Scr PTTG shRNA D Scr PBF shRNA

92-VU-040T 92-VU-040T

93-VU-147T 93-VU-147T

Supplementary Figure S5. Depletion of PTTG and PBF impairs wound healing and transforming abilities of HNSCC cells. A, Representative images of scratch wound assays in 93-VU-147T cells transduced with either PTTG or Scr shRNA. Images taken after 0 and 24 h. Scale bars, 500 m. (right) % wound recovery determined at 24 h (mean ± s.e.m., n = 3 independent experiments; unpaired two-tailed t-test; ***, P < 0.001). B, Same as (A) but instead showing wound healing in PBF shRNA transduced 93-VU-147T cells. C, Representative colony formation assay images of 92-VU-040T and 93-VU-147T cells stably transduced with either PTTG or Scr shRNA from n = 3 experiments. D, Same as (C) but cells were stably transduced with PBF shRNA. In colony formation assays 1 x 104 HNSCC

cells were seeded in 100 mm tissue culture dishes and incubated in a humidified atmosphere at 37°C and 5% CO2. After 18 days incubation colonies were fixed with 100% methanol for 15 min at RT and stained with 0.05% crystal violet for 30 min. Excess crystal violet was removed by PBS washing and dishes allowed to air-dry prior to examination of colonies. A WT p53 HNSCC TCGA; n = 157; 74/129 (P < 0.001); 106/129 genes (P < 0.05)

***P < 0.001 SESN3 CDK6 THBS1 SERPINB5 HUS1 PTEN PERP TLK1 SERPINE1 EI24 RB1 ZMAT3 RAD50 CREB1 CRY2 CCND1 CCND2 ATM GTF2H1 CD82 *P < 0.05; SFN MYC TP63 TP53I3 SESN2 WRN CDKN1A SESN1 PRKDC RRM2B **P < 0.01 NBN SMC1A MLH3 PMS2 APAF1 STEAP3 ATR BRCA2 RCHY1 GADD45A

NS SMC3 BID PPM1D MSH3 MPG CCNG1 TSC2 CCNE1 XPC XRCC2

TNFRSF10B PML UBE2A RFC1 RBM4 RAD21 BCL2 POLH RFWD2

*P < 0.05; MLH1 REV1 WRNIP1 DCLRE1A FAS MDM4 CASP2 CHEK1 CASP3 TERF1

**P < 0.01 RBBP4 TP73 CCND3 CDKN1B APEX1 CASP9 PMAIP1 POLD3 MSH2 XRCC6

PMS1 MIF RAD1 TP53 BRCA1 CDC25A GTSE1 BBC3 XRCC3

FANCD2 CCNE2 H2AFX NTHL1 CDK2 MBD4 BAX UNG RAD17 EXO1

***P < 0.001 CDKN2A OGG1 CDK4 POLE FANCG TREX1 FANCC PARP2 RRM2 DDB2

MGMT RAD9A RPA2 MUTYH CHEK2 RAD23A CHAF1A RAD51 LRDD LIG1

RAD51C FEN1 XRCC1 CDK1 POLD1 CCNB2 E2F1 CCNB1 CDC25C

Correlation ():

-0.51 0.0 0.5 0.78 B SESN3 CDK6 CDC25C E2F1 12 14 12 16 14 10 12 10 10 12 8 8 10 8 6 6 8 6 6 4 4 4 4 E2F1 Expression CDK6 Expression SESN3 Expression SESN3 2 = -0.509  = -0.474 2  = 0.777  = 0.701 2 CDC25C Expression 2 P = 1.0 x 10-11 P = 3.57 x 10-10 P = 5.16 x 10-33 P = 1.66 x 10-24 0 0 0 0 6 8 10 12 14 6 8 10 12 14 8 9 10 11 12 13 14 8 9 10 11 12 13 14 PTTG Expression PTTG Expression PTTG Expression PTTG Expression

CDBCL2 CDKN2A PPM1D SFN EI24 CCND1 *** *** *** ** ** *** Expression Expression

WT MUT WT MUT WT MUT WT MUT WT MUT WT MUT p53 p53 p53 p53 p53 p53 p53 p53 p53 p53 p53 p53

Supplementary Figure S6. Correlation of PTTG expression with p53-target genes in WT p53 HNSCC. A, Heatmap showing relative correlation values () for PTTG expression with a panel of 129 p53-target genes using TCGA data (n = 157 unmatched WT p53 HNSCC samples). Significant correlations with PTTG were observed for 82.2% (*, P <0.05,n = 106/129 genes) and 57.4% (***, P < 0.001, n = 74/129 genes) of p53-target genes according to the level of significance. P and  values were calculated using the Spearman’s correlation test. B, Representative scatterplots showing significant correlations for expression of SESN3, CDK6, CDC25C, and E2F1 (n = 157 unmatched HNSCC

samples; ***, P < 0.001; Spearman’s correlation test). C, Box whisker plots of p53-target expression (log2)with >0.45(WTp53-MUTp53) in unmatched HNSCC with either WT or MUT p53. Expression of BCL2, CDKN2A, and PPM1D was significantly lower in MUT p53 HNSCC (n = 363) compared to WT p53 HNSCC (n = 157) (***, P < 0.001; Mann-Whitney test). D, Same as (C) but showing p53-target

(log2)with <-0.3(WTp53-MUTp53) in unmatched HNSCC. Expression of SFN, EI24, and CCND1 was significantly increased in MUT p53 HNSCC (n = 363) compared to WT p53 HNSCC (n = 157) (**, P < 0.01; ***, P < 0.001; Mann-Whitney test). A MUT p53 HNSCC TCGA; n = 363; 65/129 genes (P < 0.001); 88/129 genes (P < 0.05)

ZMAT3 ATM SESN3 PPM1D CREB1 PRKDC BCL2 TLK1 WRN SMC1A

***P < 0.001 TSC2 ATR APAF1 CRY2 TP63 RRM2B CDK6 MDM2 THBS1 XPC

STEAP3 REV1 RB1 CDKN1B RBBP4 MLH3 BRCA2 RAD21 CASP2 RCHY1 *P < 0.05; **P < 0.01 SMC3 SERPINB5 CCND2 RAD50 PTEN HUS1 MDM4 NBN TP53 PERP DCLRE1A MSH3 RFWD2 PMS1 MSH2 SESN1 CASP9 CDKN2A GTF2H1 XRCC2

NS CD82 MYC BID TERF1 BRCA1 SESN2 RFC1 POLD3 TP53I3 POLH CCNE2 SERPINE1 CDKN1A CASP3 TP73 MLH1 RAD18 RAD1 TNFRSF10B UNG

PMS2 EI24 FANCD2 CCND1 FAS UBE2A GADD45A OGG1 PMAIP1 GTSE1 *P < 0.05; **P < 0.01 APEX1 CDK2 POLE CHEK1 MBD4 FANCC CCNE1 RAD17 EXO1 MGMT WRNIP1 BBC3 CHEK2 PARP2 CCNG1 SFN RRM2 XRCC6 RBM4 MUTYH

***P < 0.001 CHAF1A PML CDK4 RAD23A XRCC3 DDB2 FANCG TREX1 CDC25A XRCC1 CCND3 H2AFX RAD51C RAD9A NTHL1 LRDD RPA2 MIF LIG1 FEN1

RAD51 MPG E2F1 CDK1 BAX CCNB2 POLD1 CCNB1 CDC25C

Correlation ():

-0.53 0.0 0.64

B ATM PPM1D CCNB1 MPG 12 12 14 14 10 10 12 12 10 10 8 8 8 8 6 6 6 6 4 4 4 4

ATM Expression ATM  = -0.507  = -0.408  = 0.538  = 0.412 MPG Expression

2 PPM1D Expression 2 CCNB1 Expression 2 2 P = 3.75 x 10-25 P = 5.01 x 10-16 P = 1.15 x 10-28 P = 2.63 x 10-16 0 0 0 0 6 8 10 12 14 6 8 10 12 14 6 8 10 12 14 6 8 10 12 14 PTTG Expression PTTG Expression PTTG Expression PTTG Expression C

Supplementary Figure S7. Correlation of PTTG expression with p53-target genes in MUT p53 HNSCC. A, Heatmap showing relative correlation values () for PTTG expression with a panel of 129 p53-target genes using TCGA data (n = 363 unmatched MUT p53 HNSCC samples). Significant correlations with PTTG were observed for 68.2% (*, P < 0.05; n = 88/129 genes) and 50.4% (***, P < 0.001; n = 65/129 genes) of p53- target genes according to the level of significance. P and  values were calculated using the Spearman’s correlation test. B, Representative scatterplots showing significant correlations for expression of ATM, PPM1D, CCNB1 and MPG (n = 363 unmatched HNSCC samples; ***, P < 0.001; Spearman’s correlation test). C, Direct comparison of correlation values for PTTG expression with 129 p53-target genes in MUT p53 (upper) and WT p53 HNSCC (lower). p53 target genes are listed in alphabetical order. A WT p53 HNSCC TCGA; n = 157; 23/129 genes (P < 0.05); MUT p53; n = 363; 49/129 genes (P < 0.05)

WT p53 MUT p53 B PBF C PBF UBE2A XRCC3 1.0 WT p53 0.8 PERP MYC 0.8 MUT p53 0.6 MPG, NTHL1, H2AFX, UNG, BID MYC THBS1 )

) 0.6 SERPINB5 SERPINE1  CASP9 0.4 TP63 0.4 CCNE1 RBBP4 BID MUT p53

STEAP3  0.2

ATR 0.2 - PMS2 MPG 0 MDM4 0.0 CCND1 REV1 WT p53  ( MBD4 Correlation ( -0.2 -0.2 UBE2A, MLH1, RAD17

SERPINB5 XRCC3 UNG  RBBP4, CDKN1A -0.4 UBE2A -0.4 RBBP4 MYC CHAF1A Mean =0.071 -0.6 -0.6 SERPINE1 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 THBS1 THBS1 p53-target genes p53-target genes CCND1 CASP9 D PBF PBF MPG 1.0 WT p53 1.0 MUT p53 All genes (n = 19,764) STEAP3 p53 target genes (n = 129) 0.8 0.8 BID

0.6 0.6 Kolmogorov-Smirnov test Correlation (): 0.4 0.4

0.2 0.2 Cumulative fraction Cumulative fraction P = 0.0006 P = 9.0 x 10-6 -0.28 0.0 0.28 0 0 -0.5 0 0.5 1.0 -0.5 0 0.5 1.0 Correlation Correlation E WT p53 UBE2A STEAP3 G 92-VU-040T 93-VU-147T 14 14 12 12 PBF Scr PBF Scr shRNA 10 10 - ++--++- IR 8 8 18kDa -H2AX 6 6 53kDa p53 pS15 4 4 53kDa  = -0.272  = 0.276 p53

2 Expression STEAP3 2 UBE2A Expression UBE2A P = 5.69 x 10-4 P = 4.66 x 10-4 44kDa -actin 0 0 10 11 12 13 14 10 11 12 13 14 PBF Expression PBF Expression H 9 F MUT p53 8 * 92-VU-040 93-VU-147 Scr shRNA Scr shRNA XRCC3 THBS1 7 12 18 PBF shRNA PBF shRNA 16 6 10 14 5 8 12 * 10 4 6 8 3 NS NS * 4 6 NS NS 2 NS NS NS  = -0.246 4  = 0.252 THBS1 Expression XRCC3 Expression 2 1 P = 2.0 x 10-6 2 P = 2.42 x 10-6

0 0 –IR) vs Relative expression (+IR 0 10 11 12 13 14 15 10 11 12 13 14 15 PBF Expression PBF Expression TP53 CDKN1A BCL2 RAD51 BRCA1

Supplementary Figure S8. Association of PBF with p53-target genes. C. A, Heatmap showing relative correlation values for PBF expression with p53-target genes in WT p53 (left, n = 157) and MUT p53 (right, n = 363) unmatched HNSCC. Genes shown have  values < -0.2 or > 0.2; *, P < 0.05. B, Correlation pattern of PBF expression with 129 p53-target genes in unmatched HNSCC with WT p53 (blue dots; n = 157) and MUT p53 (red dots; n =363).C, Differences in  values for p53-target genes between WT and MUT p53 HNSCC. Genes were ordered according to increasing  value (WTp53-MUTp53; range -0.25 to +0.28). D, Cumulative distribution plot comparing the correlations between PBF with all detectable genes in TCGA (n = 19,764) versus p53-target genes (n = 129) in WT (upper) and MUT p53 (lower) HNSCC. Significant difference between distributions determined by the Kolmogorov-Smirnov test. E, Representative scatterplots showing significant correlations for expression of UBE2A and STEAP3 with PBF in WT p53 HNSCC (n = 157 unmatched HNSCC; **, P < 0.01; ***, P < 0.001; Spearman’s correlation test). F, Same as (E) but showing scatterplots for expression of XRCC3 and THBS1 with PBF in MUT p53 HNSCC (n = 363 unmatched HNSCC; ***, P < 0.001; Spearman’s correlation test). G, Western blot analysis of -H2AX, p53 S15 and p53 in PBF or Scr shRNA transduced 92-VU-040T and 93- VU-147T cells irradiated with 15-Gy dose. H, Relative fold-change in mRNA expression of indicated genes following irradiation of PBF or Scr shRNA stably transduced 92-VU-040T and 93-VU-147T cells compared with non-irradiated controls (mean ± s.e.m., n = 3 independent experiments; unpaired two-tailed t-test; ns-not significant; *, P < 0.05).

Supplementary Figure S10. PTTG and PBF influence p53 stability. A, Western blot analysis confirming successful immunoprecipitation of p53 in HNSCC cells. 1Blot probed with an anti-p53 rabbit polyclonal antibody (Santa Cruz Biotechnology). B, Reciprocal co-IP assay in 93-VU-147T cells showing specific interaction between PTTG and p53. Cell lysates were immunoprecipitated with an anti-HA antibody. C, Western blot analysis confirming successful immunoprecipitation of PBF-HA and PTTG-HA in HNSCC cells. 2Blot probed with an anti-HA rabbit polyclonal antibody (Santa Cruz Biotechnology). D, Co-IP assay in PBF shRNA stably transduced 93-VU-147T cells showing reduced interaction between PTTG and p53. (right) Mean p53 protein levels relative to -actin (mean ± s.e.m., n = 3 independent experiments; unpaired two-tailed t-test; *, P <0.05).E, Quantification of mean p53 protein levels from co-IP assays in PTTG shRNA transduced 92-VU-040T cells showing greater interaction between PBF and p53. (mean ± s.e.m., n = 3 independent experiments; unpaired two-tailed t-test; **, P <0.01).F, Western Blot analysis of p53 stability in 93-VU-147T cells transfected with VO, PBF-HA, PTTG or PBF-HA+PTTG and then lysed at indicated times post-treatment with 100 M anisomycin. (right) Quantification of mean p53 protein levels relative to -actin from 3 independent experiments at indicated time points (mean ± s.e.m., n = 3 independent experiments; unpaired two-tailed t-test; NS, not significant; **, P <0.01).H, Fold-changes in relative p53 protein levels normalised to -actin at indicated time points post-anisomycin treatment in 93-VU-147T cells transfected with VO, PTTG, PBF or PBF+PTTG . I, Fold-changes in relative p53 protein levels normalised to -actin at 120 min post-anisomycin treatment in 92-VU-040T and 93-VU-147T cells transfected with VO, PBF+PTTG, PBF+PTTG BD-, PBF M1+PTTG or PBF M1+PTTG BD- (mean., n = 3 independent experiments, **, P < 0.01; ***, P < 0.001). J, Western Blot analysis of p53 stability in 93-VU-147T cells transfected with PBF+PTTG BD-, PBF M1+PTTG or PBF M1+PTTG BD- and then lysed at indicated times post-treatment with 100 M anisomycin. K, Mean p53 protein levels normalised to -actin from half-life study described in (J). Data presented as mean ± SE from 3 independent experiments; **, P < 0.001.. A WT p53 HNSCC TCGA (n = 157)

MYC SERPINB5 BID Key *** ** PBF & PTTG PBF & PTTG * LOW LOW HIGH LOW PBFLOW & PTTGHIGH PBFHIGH & PTTGHIGH Expression

B PBF Correlation () PBF Correlation () PBF Correlation () -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 = 129) n PBF & PTTG (Q1Q2 v Q3Q4) PBF & PTTG(Q1Q4 v Q2Q3) PBF & PTTG(Q1Q3 v Q2Q4)

n = 60/129 genes; n = 16/129 genes; n = 21/129 genes; p53 target ( genes P < 0.05 P < 0.05 P < 0.05

Key: Positive correlation (P < 0.05) No correlation (P > 0.05) *WT p53 HNSCC TCGA (n = 157) Q1Q2 (n = 38); Q1Q3 (n = 43); Q1Q4 (n = 43) Negative correlation (P < 0.05) Q2Q3 (n = 42); Q2Q4 (n = 42); Q3Q4 (n = 37)

C Stratification of PBF/PTTG subgroups D WT p53/ PTTG Larynx in lymph node staging analysis (WT p53 HNSCC) Oral Cavity Hypopharynx -8 P = 8.51x10 Oropharynx (Kruskal-Wallis) WT p53 HNSCC (n = 157) E 100 PTTG (median = 10.37; range 8.36-13.07) ** PBF (median = 12.09; range 10.87-13.58) 80 ** 26/36 22/36 60

14/36 40

PTTG Expression 10/36 PTTG < 10.37 PTTG > 10.37 20 & PBF < 12.09 & PBF > 12.09 Lymph Node Stage (%) 0 N0 N1-N3 PBFLOW/ PTTGLOW PBFHIGH/ PTTGHIGH n 20 77 4 56 PBFLOW & PTTGLOW median 9.89 9.98 10.96 10.82 (Q1Q2; n = 38) (Q3Q4; n = 37) PBFHIGH & PTTGHIGH

Supplementary Figure S11. Correlation of p53-target genes with PBF in WT p53 HNSCC with high PBF/PTTG expression. A, Box-whisker plots for indicated genes (i.e. MYC, SERPINB5 and BID) in WT p53 HNSCC with high PBF/high PTTG tumoural expression (n = 37; Q3+Q4) compared to other HNSCC subgroups [(i.e. low PBF/low PTTG (n = 38; Q1+Q2), low PBF/high PTTG (n = 41) and high PBF/low PTTG (n = 41)] (***, P<0.001; **, P<0.01; ***, P<0.001; Kruskal-Wallis test). B, Correlation of PBF expression with 129 p53-target genes in unmatched WT p53 HNSCC with high PBF/PTTG (n = 38; Q3+Q4) and low PBF/PTTG expression (n = 37; Q1+Q2; 75 HNSCC samples in total). P and  values were calculated using Spearman’s correlation tests. Correlations were further determined in HNSCC with different PBF/PTTG expression subgroups, including (middle) PBF/PTTG (Q1+Q4, n = 43) and PBF/PTTG (Q2+Q3, n = 42) (85 HNSCC samples in total), as well as (right) PBF/PTTG (Q1+Q3, n = 43) and PBF/PTTG (Q2+Q4, n = 42) (85 HNSCC samples in total). C, Overview of patient stratification based on PBF and PTTG expression in WT p53 HNSCC (n = 157) for lymph node analysis (Figure 6H). Median cut off values for PTTG and

PBF in different expression subgroups are shown as well as patient numbers. D, Box whisker plot of PTTG expression (log2) at the indicated anatomical subsite in WT p53 HNSCC samples. (***, P = 8.51x10-8; Kruskal-Wallis test). Median values and number of samples are shown. E, Association of PBF/PTTG expression subgroups with lymph node staging in WT p53 HNSCC; **, P < 0.01; Fisher’s exact test. Cut-off values were adjusted for stratification according to median values shown in (D) for each different tumour subsite to correct for any potential bias.

Supplementary Figure S13. Characterisation of TP53 mutations and copy number in HNSCC TCGA. A, Graphical representation of 446 mutations in HNSCC TCGA: includes 2 duplicate mutations in patients with multiple samples. Mutation diagram circles are coloured with respect to the corresponding mutation types. In case of different mutation types at a single position, colour of the circle is determined with respect to the most frequent mutation type (cBioPortal). Mutation types are: missense (green); truncating mutations (black) – i.e. nonsense, frameshift deletion, frameshift insertion, splice site; inframe Mutations (brown) – i.e. inframe deletion, inframe insertion. B, Number of patients in HNSCC TCGA with the indicated number of TP53 mutations per tumour sample. C, Number of patients in HNSCC TCGA with a single TP53 mutation per tumour (n = 287) and the indicated TP53 alteration (i.e. missense, nonsense, frame-shift and inframe). D, Number of patients in HNSCC TCGA with indicated putative TP53 copy number (i.e. diploid. Shallow deletion, deep deletion, gain, amp) and TP53 alterations per tumour. E, Table showing number of patients in HNSCC TCGA with a single TP53 mutation per tumour and the indicated TP53 alteration and putative copy number. A B PBF: p53 target gene correlation () TP53 copy# Diploid Shallow deletion

TP73 ZMAT3 p53 missense mutations (n = 57 per group) 0.5 RRM2B CCND3 0.4 SESN1 TSC2 ) XPC  0.3 TP53 0.2 Diploid 0.1 BID Shallow deletion 0.0

-0.1 FEN1 -0.2 PARP2 Diploid PBF Correlation ( PBF Correlation -0.3 Shallow deletion XRCC6 -0.4 PMS2 MYC XRCC3 -0.5 0 20 40 60 80 100 120 140 Correlation (): p53-target genes -0.5 0.0 0.5 CDPBF PTTG

0.8 PMS2, FEN1, ZMAT3 0.8 DDB2, PARP2 0.6 0.6 ) ) WRN, CCNE2, MDM4 0.4 0.4 REV1, MDM2

0.2 0.2 DELETION DELETION  

- 0.0 - 0.0

-0.2 -0.2 DIPLOID DIPLOID TP53, TSC2, RFC1   ( -0.4 PMAIP1, RCHY1, RRM2B ( -0.4 MGMT, MLH1

 SESN1, XPC  -0.6 -0.6 Mean = 0.171 Mean = 0.129 -0.8 -0.8 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 p53-target genes p53-target genes

Supplementary Figure S14. Influence of TP53 genomic copy number on correlation of PBF and PTTG with p53-target genes. A, Correlation pattern of PBF with 129 p53-target genes in unmatched HNSCC with MUT p53 (single missense mutation per tumour) and either diploid (blue dots) or shallow deletion for TP53 (red dots) (n = 57 per group). B, Heatmap showing relative correlation values () for PBF with top ten most correlated p53-target genes (i.e. upper, positively correlated genes; lower, negatively correlated genes) in HNSCC TCGA with diploid versus shallow deletion for TP53 copy number. C, Differences in  values for PBF and p53-target genes in MUT p53 HNSCC with a single missense mutation and different TP53 copy number (i.e. diploid versus shallow deletion; n = 57 per group). Genes ordered according

to increasing  value (DIPLOID-DELETION; range -0.41 to +0.54). D, Differences in  values for PTTG and p53-target genes in MUT p53 HNSCC with a single missense mutation and different TP53 copy number (i.e. diploid versus shallow deletion; n = 57 per group). Genes

ordered according to increasing  value (DIPLOID-DELETION; range -0.59 to +0.38). The genomic copy number loss of wild-type TP53 disrupts the transcriptional relationship between PBF and PTTG with p53-target genes in tumour cells.