Nature Genetics: doi: /ng Supplementary Figure 1. TCGA data set on HNSCCs reanalyzed in this study.

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1 Supplementary Figure 1 TCGA data set on HNSCCs reanalyzed in this study. Summary of the TCGA dataset on HNSCCs re-analyzed in this study and the respective numbers of samples available within each.

2 Supplementary Figure 2 The methylation-defined H3 K36 subgroup consists of samples carrying damaging NSD1 or H3 K36M alterations. (a) Our analysis reveals that the H3K36 methylation cluster (Fig. 1) is comprised of 61 samples. Ten samples carry H3K36M alterations, while 51 samples have NSD1 alterations, with one carrying both. One sample had no available genomic/transcriptome data. (b) H3K36M alteraions are observed in a wide array of histone H3 genes, supporting the dominant nature of this K-to-M substitution as reported 32. (c) Samples in the H3K36 cluster are enriched for nonsense (in red) NSD1 mutations. Missense mutations (in green) are often observed near or at the functional SET domain.

3 Supplementary Figure 3 IGV screenshots of complex genomic events in NSD1 using RNA-seq data. (a) A focal deletion of a set of 10 exons in sample BA (b) Near absent expression of NSD1 in sample CN (c) Aberrant NSD1 splicing, possibly due to a translocation in sample D (d) A Focal deletion of a set of 14 exons in sample UF-A7JH

4 Supplementary Figure 4 Heat map showing hierarchical clustering of HNSCC samples based on DNA methylation and genetic alterations in other genes associated with HNSSC. (Top) Previous 4-class, expression-based classification (Atypical (black), Classical (red), Mesenchymal (green), Basal(blue)). (Middle) Other genes involved in the H3K36 methylation pathways are infrequently mutated in HNSCC and do not vary within HPV- HNSCC subgroups. (Bottom) Only TP53, CASP8, NSD1 and H3K36M vary significantly among HPV- HNSCC subgroups. For color coding of methylation groups and anatomical locations, please see Figure 1.

5 Supplementary Figure 5 Mean sample methylation levels of HNSCC. Mean sample methylation levels of HNSCC subgroups showing that NSD1 and H3K36M alterations are similarly associated with a DNA hypomethylated phenotype compared to the other four methylation clusters identified in HNSCCs.

6 Supplementary Figure 6 Global DNA methylation, NSD1 expression level and the types of mutations in the H3 K36M cluster. (a,b) Samples with NSD1 mutations outside the H3K36M cluster (grey bars) show higher global DNA methylation levels (a) and higher NSD1 gene expression levels (b) compared to samples in the H3K36 cluster (blue bars). (c) Samples in the H3K36 cluster frequently carry NSD1 truncating (nonsense and frameshift) mutations compared to NSD1 mutated samples in the other HNSCC subgroups.

7 Supplementary Figure 7 Mean somatic mutations per sample of HNSCC subgroups. Mean somatic mutations per sample of HNSCC subgroups showing that NSD1 mutant samples have higher somatic mutation count than H3K36M samples or other HNSCC subgroups.

8 Supplementary Figure 8 NSD1 but not H3 K36M mutant samples have smoking-induced somatic mutation signatures. (a) In NSD1 samples, similar to lung adenocarcinoma (LUAD) and lung squamous cell carcinomas (LUSC), a large proportion of somatic mutations is accounted by the smoking-associated signature S4 (red). In comparison, other HNSCC samples (left) have much less smoking-induced somatic mutations. (b) Unsupervised clustering of mutation frequencies groups NSD1, but not H3K36M, with lung cancers, suggesting the mutational spectrum in NSD1 samples is associated with smoking. (c) A five-signature model identifies UV exposure (S1), Temozolomide (S2) and smoking (S4) as major mutation generation processes.

9 Supplementary Figure 9 Kaplan Meier curve for HNSCC patients, grouped by DNA-methylation-defined molecular subgroups.

10 Supplementary Figure 10 Anatomical distribution of HNSCC samples relative to K36M and NSD1 alterations. Anatomical distribution of HNSCC samples showing that H3K36M samples are exclusively found in the oral cavity whereas the majority of NSD1 mutated samples are found in the larynx and oral cavity.

11 Supplementary Figure 11 NSD1 and NSD2 expression in the H3K36 cluster and other HNSCC subgroups (a) Samples in the H3K36 cluster show lower NSD1 expression levels. (b) NSD2 levels are uniform among al HNSCC subgroups.

12 Supplementary Figure 12 RNA-expression clustering of HNSCC samples. Similar to our findings using DNA methylation, we observe a group of samples enriched in NSD1/H3K36M alterations (H3K36 cluster) and another enriched for HPV+ HNSCC. For color coding of methylation groups and anatomical locations, please see Figure 1.

13 Supplementary Figure 13 Gene ontology enrichment analysis on H3K36-specific differentially expressed genes. Differential expression analysis performed on H3K36 samples versus all other HNSCCs tumors, controlling for anatomical location.

14 Supplementary Figure 14 Western blots for H3 K36M and NSD1 in HNSSC cell lines. Full length immunoblots including molecular weights that were used in Figure 4.

15 Supplementary Table 1. NSD1 mutations in samples included in the H3K36 cluster. Hugo Symbol NSD1, Chromosome 5. Sample ID Protein Mutation Start End Position Reference Variant Allele Mutation Change Type Position Allele Allele Frequency Assessor TCGA-BA A-01D p.r788x Nonsense C T TCGA-CV A-01D p.h1872fs Frame_Shift_Del AT TCGA-CV A-31D p.q1153x Nonsense C T NA TCGA-BB A-11D p.e990x Nonsense G T TCGA-BA A-11D p.s707x Nonsense C G TCGA-CV A-41D p.g1678w Missense G T Medium TCGA-CV A-11D p.e1534x Nonsense G T 0.75 TCGA-CR A-11D p.g1524fs Frame_Shift_Del G TCGA-CR A-11D p.p2567t Missense C A 0.1 Neutral TCGA-CR A-11D p.q1989x Nonsense C T TCGA-CR A-11D p.t723s Missense A T Neutral TCGA-CR A-11D p.e1979x Nonsense G T TCGA-CV A-11D p.s1086fs Frame_Shift_Del AGTAAAG TCGA-CR A-11D p.s1528fs Frameshift Insertion A TCGA-CV A-11D p.s1359x Nonsense C A TCGA-CV A-11D p.a2009t Missense G A High TCGA-CR A-01D p.y1997c Missense A G High TCGA-CN A-01D p.g1928x Nonsense G T TCGA-CN A-01D p.g959x Nonsense G T TCGA-CN A-01D p.s589fs Frame_Shift_Del TT TCGA-CN A-01D p.k1433x Nonsense A T TCGA-F A-11D p.m1094fs Frame_Shift_Del G TCGA-CV A-11D p.c1619s Missense G C Medium TCGA-CV A-11D p.c792x Nonsense C A TCGA-CV A-11D p.r1473x Nonsense C T TCGA-CV-A461-01A-41D-A25Y-08 p.r1757fs Frame_Shift_Del G TCGA-CV-A461-01A-41D-A25Y-08 p.s981fs frameshift insertion G TCGA-BA-A4IF-01A-11D-A25Y-08 p.p1665l Missense C T Medium TCGA-CN-A497-01A-11D-A24D-08 p.e1391x Nonsense G T TCGA-CN-A497-01A-11D-A24D-08 p.w2032x Nonsense G A TCGA-CN-A63W-01A-11D-A30E-08 p.w1160x Nonsense G A Note Sample also has NSD1 frame shift deletion and nonsense mutation Sample also has NSD1 frame shift deletion and nonsense mutation Sample also has NSD1 nonsense mutation TCGA-CN-A641-01A-11D-A30E-08 p.c1897f Missense G T High Sample also has NSD1 frame shift insertion TCGA-CN-A641-01A-11D-A30E-08 p.i1946fs frameshift insertion T TCGA-CN-A641-01A-11D-A30E-08 p.r1948i Missense CG AT Medium Sample also has NSD1 frame shift insertion TCGA-CN-A63T-01A-11D-A28R-08 Splice G A 0.2 TCGA-CN-A63T-01A-11D-A28R-08 p.e1970a Missense A C High Sample also has NSD1 splicing mutation TCGA-BB-A5HZ-01A-21D-A28R-08 p.g1095fs Frame_Shift_Del GCCACTTAA TCGA-BB-A5HZ-01A-21D-A28R-08 p.i1291fs Frameshift Insertion T 0.2 TCGA-P3-A5QF-01A-11D-A28R-08 p.r2005q Missense G A Medium TCGA-KU-A66S-01A-21D-A30E-08 p.h1616y Missense C T Medium Sample also has NSD1 frame shift deletion TCGA-KU-A66S-01A-21D-A30E-08 p.r1952fs Frame_Shift_Del G TCGA-CN-A63U-01A-11D-A30E-08 p.e1501x Nonsense G T TCGA-CQ A-21D-A30E-08 p.q679x Nonsense C T TCGA-BA-A6DL-01A-21D-A30E-08 p.r1072x Nonsense C T TCGA-BA-A6DL-01A-21D-A30E-08 p.r1200fs Frame_Shift_Del GGGATGAG TCGA-H7-A6C5-01A-11D-A30E-08 p.1664_1665nonframe_shift_del TCC TCGA-D6-A6EO-01A-11D-A31L-08 p.e2028fs Frame_Shift_Del A TCGA-D6-A74Q-01A-11D-A34J-08 p.p753fs Frame_Shift_Del A TCGA-CN-A6V3-01A-12D-A34J-08 p.c1710y Missense G A High TCGA-CN-A6V3-01A-12D-A34J-08 p.w1769c Missense G T 0.45 Medium TCGA-UF-A71D-01A-12D-A34J-08 p.t922fs Frame_Shift_Del C TCGA-BA-A6DE-01A-22D-A31L-08 p.e1575x Nonsense G T TCGA-CV-A6JU-01A-11D-A31L-08 p.y1941fs Frameshift Insertion T TCGA-QK-A6VB-01A-12D-A34J-08 p.y1834fs Frameshift Insertion A TCGA-P3-A6T8-01A-11D-A34J-08 p.s2664fs Frame_Shift_Del T TCGA-UF-A719-01A-12D-A34J-08 p.s1528y Missense C A 0.7 Low Sample also has H3K36M mutation TCGA-UF-A7JF-01A-11D-A34J-08 p.e1853x Nonsense G T TCGA-QK-A8Z8-01A-11D-A p.h1872fs Frame_Shift_Del AT

16 Supplementary Table 2. Complex genomic events in H3K36 samples with no NSD1 or H3K36M mutations by Whole Exome Sequencing. LOH=Loss Of Heterozygosity; NA=Not Available Sample Result GISTIC NSD1 GISTIC NSD1 Gene Value Gene Threshold CV-7091 LOH CN-4731 Focal Deletion of 8 exons D Splicing defect CN-6988 LOH BA-4074 No expression NA NA CV-7428 Not sequenced CV-7435 No expression UF-A7JH Focal deletion exons

17 Supplementary Table 3. NSD1 mutations in samples excluded from the H3K36 cluster. Hugo Symbol NSD1, Chromosome 5. Sample ID Protein Start End Reference Variant Allele Mutation Mutation Type Change Position Position Allele Allele Frequency Assessor TCGA-CN A-02D p.c1710s Missense G C High TCGA-HD A-11D p.e1202fs Frame_Shift_Del G - NA TCGA-DQ A-11D p.p803fs Frame_Shift_Del C - NA TCGA-CR A-11D p.d1489n Missense G A Neutral TCGA-CR A-11D p.l2054r Missense T G NA High TCGA-CV A-21D p.e2467q Missense G C Low TCGA-CV A-21D p.r1984x Nonsense C T TCGA-CR A-11D p.r1700x Nonsense C T TCGA-CV A-11D p.d1992h Missense G C Neutral TCGA-CV A-11D p.d2002h Missense G C Medium TCGA-CN A-01D p.s744x Nonsense C G TCGA-CV A-11D p.i2113m Missense C G Low TCGA-CV A-11D p.d325v Missense A T Medium TCGA-CV A-21D p.s96c Missense C G Neutral TCGA-CV A-11D p.p434l Missense C T Low TCGA-CV A-11D p.p434s Missense C T Low TCGA-D A-11D p.v366l Missense G T Low TCGA-CV-A468-01A-11D-A25Y-08 p.r1320x Nonsense C T TCGA-CV-A45Z-01A-21D-A25D-08 p.k601x Nonsense A T TCGA-F7-A623-01A-11D-A28R-08 p.e1520k Missense G A Low TCGA-UF-A71B-01A-12D-A34J-08 p.g566e Missense G A Neutral TCGA-TN-A7HL-01A-11D-A34J-08 p.r1634q Missense G A Medium TCGA-P3-A6T6-01A-11D-A34J-08 p.e1516x Nonsense G T TCGA-QK-AA3J-01A-11D-A p.r788x Nonsense C T

18 Supplementary Table 4. H3K36M Mutations in TCGA HNSCCs Hugo Protein Start End Reference Variant Allele Sample ID Mutation Type Symbol Change Chromosome Position Position Allele Allele Frequency H3F3B TCGA-CQ A-11D p.k37m* nonsynonymous SNV T A H3F3B TCGA-QK-A8Z7-01A-11D-A p.k37m nonsynonymous SNV T A HIST1H3C TCGA-CN A-01D p.k37m nonsynonymous SNV A T HIST1H3C TCGA-CQ A-11D p.k37m nonsynonymous SNV A T HIST1H3C TCGA-CQ-A4C9-01A-11D-A25D-08 p.k37m nonsynonymous SNV A T HIST1H3C TCGA-MT-A67D-01A-31D-A30E-08 p.k37m nonsynonymous SNV A T HIST1H3E TCGA-CV A-11D p.k37m nonsynonymous SNV A T HIST1H3G TCGA-UF-A7JD-01A-11D-A34J-08 p.k37m nonsynonymous SNV T A HIST1H3I TCGA-CV A-11D p.k37m nonsynonymous SNV T A HIST1H3I TCGA-UF-A719-01A-12D-A34J-08 p.k37m nonsynonymous SNV T A HIST2H3D TCGA-CX A-11D p.k37m nonsynonymous SNV T A *K37M is the universal nomenclature when describing amino acid changes in genomic data. We note that histone proteins have the first methionine removed from the final product thus shifting the numbering by one amino acid. In the context of protein sequences, this mutation will generally be referred to as K36M.

19 Supplementary Table 6. Meta analysis of previous HNSCC sequencing studies reveals other NSD1 and H3K36M mutations. Table adapted from Riaz, Morris, Lee, & Chan. Unraveling the molecular genetics of head and neck cancer through genome-wide approaches. Genes Dis 1; 75-86, 2014 Sequencing Study (# Samples Sequenced) Stransky (74) NSD1 p.c1710* (Larynx, Smoker, HPV-), p.c2124y (Larynx, Smoker, HPV-), p.p1726h (Larynx, Smoker, HPV-), p.r1233fs (Larynx, Smoker, HPV-), p.r1984q (Hypopharynx, Smoker, HPV-), p.y1834fs (Larynx, Smoker, HPV-), splicing (Oral Cavity, Smoker, HPV-) H3K36M HIST1H3H (Oral Cavity, Smoker, HPV-) Agrawal (32) 0 0 Pickering (40) p.r2017q (Tongue, Smoker, HPV-), p.w2032r (FOM, Smoker, HPV-), 0 p.r1984x (FOM, Smoker, HPV-) India ICGC (50) p.q517* (Gingivo-buccal oral, NA, NA), p.s958l (Gingivo-buccal oral, NA, NA), p.r1811* (Gingivo-buccal oral, NA, NA), p.c1911f (Gingivo-buccal oral, NA, NA) 0