Supplementary tables
Supplementary Table 1 Clinical data and related experiments
Experiment Number Exome target capture
Gender Age Tumour location Other
type of cases sequencing
a low tumour mutation burden (TMB)-1
The patient had an irregular mass at the
mutation/Mb; PTEN (p.C71fs*3;
Single cell tongue base, approximately 5.1×4.8 cm in
1 Female 37 Tongue D24fs*20), KMT2D (p.G2493V),
sequencing size, with unclear margins and multiple
PDGFRA (p.T281M), STK11 (p.354L),
cervical lymph node metastases
and CTNNA1 (p.D265E) mutations
The primary site was epiglottis ACC.
Radiotherapy (60 GY) was applied after
TMB-0.4 mutations/Mb; KDM6A
Single cell resection of the primary tumour, and
1 Female 58 Lung metastases (p.Q191X) and TP53 (c.920-5C>T)
sequencing surgical treatment was performed for
mutations
pulmonary metastasis in the fifth year after
surgery
ChIP-seq 1 Female 33 Maxillary sinus MYB-NFIB gene fusion mutation MYB-ChIP Seq
Nasal sinus (13 cases), salivary
Male (16
21-71 gland (3 cases), larynx (2
cases),
Immunohistoc (Median cases), bronchus (2 cases),
34 female Detection of MYB, NICD1
hemistry age: lacrimal gland (3 cases),
(18
43.5) external auditory canal (3 cases)
cases)
and other parts (8 cases)
Male (14
cases), 25-69
Immunohistoc Nasal sinus (9 cases), lung Detection of MYB, NICD1, RARα, RARβ,
37 female (Median
hemistry metastasis (28cases) RARγ, FABP7, NPM1, PRMT3
(23 age: 53)
cases)
Supplementary Table 2 Cell type definition based on signature genes and their distribution ratio in tissues
Cluster Cell type Canonical markers Literature Cluster Ratio in primary tissue or lung metastases
AP A F A1 B1 C1
Maria OM et al. 2012;
0 Serous acinar cell EFHD1D, LTF, PRR4 Plasschaert LW et al, 0.000 0.419 0.000 0.558 0.100 0.174
2018
1 Mucous acinar cell BMP6, ALCAM, MUC5B Maria OM et al. 2012 0.000 0.008 0.000 0.194 0.126 0.462
2 Acinar cell BMP6, SCGB3A2, Heikinheimo KA,1999 0.000 0.004 0.002 0.021 0.494 0.033
3 Myoepithelial cell TP63, MYL9 Bilal et al. 2003 0.000 0.092 0.000 0.099 0.078 0.126
Finkelstein et al. 1995;
4 CD8+ T cell CD3E, CD8A, PDCD1, CTLA4 0.068 0.051 0.232 0.037 0.032 0.041
Banat et al. 2015 (k, i)
5 Fibroblast FN1, DCN, COL1A1, PDGFRA Crapo et al. 1982 0.490 0.002 0.001 0.000 0.000 0.000
6 Vein endothelial cell ACKR1, VWF, PECAM1, ENG Townsley et al. 2012 0.043 0.040 0.151 0.009 0.021 0.031
7 NK cell KLRF1, KLRD1 Marquardt et al. 2017 0.109 0.067 0.026 0.005 0.056 0.008
Crapo et al. 1982;
8 Macrophage CD4, CD14, CD68, CD1C, CLEC10 Fehrenbach et al. 1994 0.001 0.002 0.209 0.002 0.002 0.003
(f)
9 Fibroblast FN1; DCN; COL1A1, PDGFRA Crapo et al. 1982 0.006 0.200 0.018 0.003 0.007 0.004
10 Unknown epithelial cell EPCAM, STMN1, MKI67, TOP2A Williams DW et al.2021 0.001 0.014 0.000 0.039 0.022 0.057
Crapo et al. 1982;
11 Alveolar type II cell SFTPB, SFTPD, MUC1 0.000 0.000 0.001 0.023 0.033 0.044
Fehrenbach et al. 1994
12 Basal cell KRT5, SFN Boers et al. 1998 0.153 0.004 0.000 0.000 0.000 0.000
Finkelstein et al. 1995;
13 Neutrophil S100A8, S100A9, IFITM2 0.003 0.000 0.091 0.001 0.001 0.003
Banat et al. 2015
14 Alveolar type I cell AGER, CLIC5 Crapo et al. 1982 (f) 0.000 0.000 0.076 0.001 0.001 0.001
Finkelstein et al. 1995;
15 Mast cell MS4A2, CPA3, TPSAB1 0.002 0.000 0.064 0.004 0.002 0.003
Banat et al. 2015
16 Dendritic cell FABP4, APOC1, C1QA Townsley et al. 2012 0.041 0.025 0.005 0.003 0.005 0.004
Vascular smooth muscle Crapo et al. 1982;
17 ACTA2, MYL9, RGS5 0.000 0.000 0.056 0.000 0.000 0.000
cell Fehrenbach et al. 1994
18 Fibroblast FN1, DCN, COL1A1, COL1A2 Crapo et al. 1982 0.057 0.000 0.012 0.001 0.001 0.001
Crapo et al. 1982;
19 Alveolar type II cell SFTPB, SFTPD, MUC1 Fehrenbach et al. 1994 0.000 0.000 0.026 0.000 0.017 0.001
(f)
Kambouchner et al.
20 Lymphatic endothelial cell PECAM1, PROX1 0.005 0.057 0.000 0.000 0.000 0.000
2009; Sozio et al. 2012
21 Ciliated epithelial cell FOXJ1, CCDC78 Crapo et al. 1982 (f) 0.000 0.001 0.018 0.003 0.001 0.003
22 Plasma cell CD79A, CD27, SLAMF7 Banat et al. 2015 (k) 0.000 0.004 0.012 0.000 0.003 0.003
Finkelstein et al. 1995;
23 B cell CD79A, MS4A1 0.022 0.004 0.000 0.000 0.000 0.000
Banat et al. 2015 (k)
24 Undefined cell 0.000 0.006 0.000 0.000 0.000 0.000
Supplementary Table 3 MYB-ChIP data region upstream of 2 kb in a patient with
MYB-NFIB fusion
chr peak start peak end gene id symbol
chr1 2627219 2627313 100287898 TTC34
chr1 29567178 29567357 10076 PTPRU
chr1 43094751 43094904 728621 CCDC30
chr1 56996120 56996262 8613 PPAP2B
chr1 63733146 63733265 199899 LINC00466
chr1 93197142 93197256 7813 EVI5
chr1 145277189 145277332 100288142 LOC100288142
chr1 145277189 145277332 388677 NOTCH2NL
chr1 145277189 145277332 400818, (100132406)
chr1 145352395 145352464 100132406 NBPF10
chr1 145352395 145352464 100288142 LOC100288142
chr1 145352395 145352464 101929780 LOC101929780
chr1 145352395 145352464 400818, (100132406)
chr1 184799385 184799534 116496 FAM129A
chr1 208412343 208412488 5362 PLXNA2
chr1 225775101 225775179 55740 ENAH
chr10 24762815 24762981 56243 KIAA1217
chr10 74454859 74455034 90550 MCU
chr10 90145709 90145965 55328 RNLS
chr10 104104110 104104261 8729 GBF1
chr10 104536760 104536935 54838 WBP1L
chr10 104756955 104757116 54805 CNNM2
chr11 19996677 19996832 89797 NAV2
chr11 20489389 20489540 10196 PRMT3
chr11 44146959 44147102 2132 EXT2
chr11 84539600 84539746 1740 DLG2
chr11 93885455 93885602 24145 PANX1
chr11 106705859 106706023 2977 GUCY1A2
chr11 126489666 126489779 84623 KIRREL3
chr11 133797470 133797607 22997 IGSF9B
chr12 21597996 21598136 79912 PYROXD1
chr13 26540878 26541017 51761 ATP8A2
chr13 35187301 35187448 100874179 LINC00457
chr13 41638436 41638567 11193 WBP4
chr13 78314454 78314597 122060 SLAIN1
chr13 113389755 113389902 23250 ATP11A
chr14 23895403 23895536 4625 MYH7
chr14 76242716 76242832 23093 TTLL5
chr14 89673045 89673161 1112 FOXN3
chr14 94689072 94689192 57718 PPP4R4
chr15 22568624 22568790 646396 REREP3
chr15 35835687 35835831 89978 DPH6
chr15 42247028 42247169 30844 EHD4
chr15 57183340 57183510 145783 LOC145783
chr15 58947500 58947648 102 ADAM10
chr15 61148258 61148395 6095 RORA
chr15 80742868 80743013 9915 ARNT2
chr15 89846643 89846764 55215 FANCI
chr16 20817823 20818082 81691 LOC81691
chr16 58556466 58556590 23019 CNOT1
chr16 77379580 77379722 170692 ADAMTS18
chr16 84091117 84091241 8720 MBTPS1
chr17 3496656 3496793 7442 TRPV1
chr17 39093738 39093859 25984 KRT23
chr17 61556437 61556587 1636 ACE
chr17 62559818 62559958 64750 SMURF2
chr18 4271874 4272014 284215 DLGAP1-AS5
chr18 4271874 4272014 9229 DLGAP1
chr18 5238788 5238913 339290 LINC00667
chr18 21362008 21362165 3909 LAMA3
chr19 8371212 8371412 51293 CD320
chr19 9213824 9213943 390882 OR7G2
chr19 12035886 12036010 90592 ZNF700
chr19 36505910 36506057 25999 CLIP3
chr19 46143941 46144083 24139 EML2
chr19 50811091 50811231 79784 MYH14
chr19 52478105 52478242 101669766 HCCAT3
chr19 52478105 52478242 59348 ZNF350
chr2 21912650 21912794 645949 LOC645949
chr2 26562518 26562709 165082 GPR113
chr2 56487010 56487151 114800 CCDC85A
chr2 74294044 74294178 200424 TET3
chr2 122179571 122179675 23332 CLASP1
chr2 131126053 131126201 26469 PTPN18
chr2 168091132 168091278 129446 XIRP2
chr2 173305237 173305367 3655 ITGA6
chr2 174027791 174027932 51776 ZAK
chr2 212402148 212402282 2066 ERBB4
chr2 231305936 231306079 6672 SP100
chr2 234294309 234294452 8527 DGKD
chr20 13699401 13699543 51575 ESF1
chr20 33323918 33324064 23054 NCOA6
chr21 11049910 11050196 85316 BAGE5
chr21 11049910 11050196 85317 BAGE4
chr21 11049910 11050196 85318 BAGE3
chr21 11049910 11050196 85319 BAGE2
chr21 34813911 34814057 757 TMEM50B
chr21 41426412 41426522 1826 DSCAM
chr21 42581288 42581436 25825 BACE2
chr22 36725581 36725742 4627 MYH9
chr22 40994555 40994695 57591 MKL1
chr3 3201409 3201543 51185 CRBN
chr3 4467685 4467820 285362 SUMF1
chr3 10260367 10260496 3656 IRAK2
chr3 10541852 10542003 491 ATP2B2
chr3 15711443 15711592 23243 ANKRD28
chr3 71098319 71098500 27086 FOXP1
chr3 73560874 73561122 23024 PDZRN3
chr3 85843181 85843325 253559 CADM2
chr3 118642146 118642279 152404 IGSF11
chr3 124259811 124259952 8997 KALRN
chr3 177163367 177163517 100505566 LINC00578
chr3 194943488 194943642 152002 XXYLT1
chr3 196946844 196946967 1739 DLG1
chr4 41122956 41123156 323 APBB2
chr4 165568813 165568956 100847071 MIR5684
chr5 65012331 65012480 54557 SGTB
chr5 76579784 76579950 8622 PDE8B
chr5 83536371 83536508 10085 EDIL3
chr5 90407373 90407612 84059 GPR98
chr5 100163542 100163692 7903 ST8SIA4
chr5 138390135 138390278 64374 SIL1
chr6 3106742 3106873 8737 RIPK1
chr6 20842174 20842365 54901 CDKAL1
chr6 69354077 69354206 577 BAI3
chr6 71548654 71548811 60682 SMAP1
chr6 75953350 75953585 1347 COX7A2
chr6 109242827 109243096 101929716 ARMC2-AS1
chr6 109242827 109243096 84071 ARMC2
chr6 136979849 136979988 4217 MAP3K5
chr6 151197823 151197968 25902 MTHFD1L
chr7 14309220 14309385 1607 DGKB
chr7 87837953 87838083 6717 SRI
chr7 90820544 90820684 5218 CDK14
chr7 92268945 92269293 1021 CDK6
chr7 116411298 116411444 4233 MET
chr7 144478675 144478820 27010 TPK1
chr7 151999562 151999684 58508 KMT2C
chr8 8743560 8743703 9258 MFHAS1
chr8 17173353 17173494 9108 MTMR7
chr8 22201404 22201546 55124 PIWIL2
chr8 39539842 39539979 8749 ADAM18
chr8 71556906 71557027 286190 LOC286190
chr8 71556906 71557027 51110 LACTB2
chr8 103420585 103420736 51366 UBR5
chr9 2179762 2179886 6595 SMARCA2
chr9 9455397 9455531 5789 PTPRD
chr9 87326945 87327140 4915 NTRK2
chr9 138848056 138848201 10422 UBAC1
chrUn_gl000220 117825 125419 100507412 LOC100507412
chrUn_gl000220 117825 125419 100861532 RNA45S5
chrX 68909990 68910119 1896 EDA
chrX 84558578 84558730 79983 POF1B
chrX 101870495 101870642 100528062 ARMCX5-GPRASP2
chrX 111274449 111274574 7224 TRPC5
chrX 117666245 117666377 139818 DOCK11
chrX 118117459 118117599 79836 LONRF3
chrX 123026386 123026518 331 XIAP
chrX 123614903 123615040 10178 TENM1
chrX 138721846 138721996 4168 MCF2
chrX 153227431 153227576 3054 HCFC1
Supplementary Table 4 Summary of materials used in the experiments
Experiment Antibodies Source Concentration Identifier
anti-c-Myb Abcam, Cat# ab45150 1:100 AB_778878
anti-cleaved Notch1 Cell Signaling Technology, Cat# 4147 1:80 AB_2153348
anti-RARα Proteintech, Cat# 10331-1-AP 1:100 AB_2177742
Immunohistochemical anti-RARβ Abcam, Cat# ab124701 1:100 AB_10975008
anti-RARγ Cell Signaling Technology, Cat# 8965 1:100 AB_10998934
anti-FABP7 Proteintech, Cat# 14836-1-AP 1:100 AB_2100458
anti-PRMT3 Abcam, Cat# ab191562 1:100 NA
anti-NPM Cell Signaling Technology, Cat# 3542 1:100 AB_2155178
anti-GAPDH Proteintech, Cat# 60004-1-Ig 1:5000 AB_2107436
anti-β-Actin Abcam, Cat# AC026 1:5000 AB_2768234
anti-c-Myb Cell Signaling Technology, Cat# 12319 1:1000 AB_2716637
anti-cleaved Notch1 Cell Signaling Technology, Cat# 4147 1:1000 AB_2153348
anti-c-MYC Proteintech, Cat# 10828-1-AP 1:1000 AB_2148585
Western blot
anti-MAZ Proteintech, Cat#21068-1-AP 1:1000 AB_2878805
anti-HES1 Cell Signaling Technology, Cat#11988 1:1000 AB_2728766
anti-RARα Proteintech, Cat# 10331-1-AP 1:1000 AB_2177742
anti-RARβ Abcam, Cat# ab124701 1:1000 AB_10975008
anti-RARγ Cell Signaling Technology, Cat# 8965 1:1000 AB_10998934
anti-cleaved Notch1 Cell Signaling Technology, Cat# 2421 1:1000 AB_2314204
Co-immunoprecipitation
anti-c-Myb Cell Signaling Technology, Cat# 12319 1:1000 AB_2716637
anti-c-MYC Proteintech, Cat# 10828-1-AP 1:200 AB_2148585
Normal Rabbit IgG Cell Signaling Technology Cat#2729S 1:200 AB_1031062
ATRA MedChemExpress, HY-14649 Cell:1 μM; Animal:5-10mg/kg
DAPT MedChemExpress, HY-13027 Cell: 20 μM; Animal:10mg/kg
Chemicals GW9662 MedChemExpress, HY-16578 1 μM
AGN193109 MedChemExpress, HY-u00449 1 μM
PV two-step IHC kit ZSGB-BIO, PV-9000
Leica light microscope Germany, DM6
TM
Lipofectamine 8000 Beyotime, C0533
2100 Bioanalyzer Agilent
Instruments In vivo image system PerkinElmer, Lumina II
CCK8 kit Applygen, E1008-200
24-well cell culture inserts Corning,3422
RNA extraction kit Yishan, ES-RN001
Reverse transcription kit Takara, RR037A
Protein A/G Agarose Sigma, P5641
T4 DNA Ligase Thermo, EL0011
penicillin–streptomycin
Biosharp, BL505A
solution
Other pCDHO-neo-MYB-3-Flag Vigene, CH898248
pcDNA3.1-MYC-HA plasmid ChemicalBook
T4 ligase Thermo, EL0011
E. coli Thermo, FD0274
293T cells CCLV, Cat# CCLV-RIE 1018
Supplementary Table 5 sh primers used in knockdown experiments
Name sh Primer Sequence
shRNA-MYB-1-F：5'-CCGGAACAGAATGGAACAGATGACCTCGAGGTCATCTGTTCCATTCTGTTCTTTTT-3'
MYB shRNA-MYB-1-R：5'-AATTAAAAAAACAGAATGGAACAGATGACCTCGAGGTCATCTGTTCCATTCTGTTC-3'
shRNA-MYB-2-F：5'-CCGGCCAGATTGTAAATGCTCATTTCTCGAGAAATGAGCATTTACAATCTGGTTTTT-3'
shRNA-MYB-2-R：5'-AATTAAAAACCAGATTGTAAATGCTCATTTCTCGAGAAATGAGCATTTACAATCTGG-3'
shRNA-NOTCH1-1-F: 5'-CCGGAGGTCAGTGTGGAGGTGGATTCTCGAGAATCCACCTCCACACTGACCTTTTTTT-3'
NOTCH1 shRNA-NOTCH1-1-R: 5'-AATTAAAAAAGGTCAGTGTGGAGGTGGATTCTCGAGAATCCACCTCCACACTGACCTT-3'
shRNA-NOTCH1-2-F: 5'-CCGGGCGCTGATCAGCGGTATTGAACTCGAGTTCAATACCGCTGATCAGCGCTTTTTT-3'
shRNA-NOCTH1-2-R: 5'-AATTAAAAAGCGCTGATCAGCGGTATTGAACTCGAGTTCAATACCGCTGATCAGCGCT-3'
shRNA-MYC-1-F：5'-CCGGCCTGAGACAGATCAGCAACAACTCGAGTTGTTGCTGATCTGTCTCAGGTTTTT-3'
shRNA-MYC-1-R：5'-AATTAAAAACCTGAGACAGATCAGCAACAACTCGAGTTGTTGCTGATCTGTCTCAGG-3'
MYC shRNA-MYC-2-F：5'-CCGGGCTTCACCAACAGGAACTATGCTCGAGCATAGTTCCTGTTGGTGAAGCTTTTT-3
shRNA-MYC-2-R：5'-AATTAAAAAGCTTCACCAACAGGAACTATGCTCGAGGTCATCTGTTCCATTCTGTTC-3'
Supplementary Table 6 Primers for qPCR
Name Primer Sequence
F：5’- GGCGAGCCCCTTGCA-3’
MYB
R：5’-CTCCTCCATCTTTCCACAGGAT-3’
F：5′-GGTGAGACCTGCCTGAATG-3′
NOTCH1
R：5′-GTTGGGGTCCTGGCATC-3′
F：5’-TGCTCCATGAGGAGACACC-3′
MYC
R：5’-CTCTGACCTTTTGCCAGGAG-3′
F：5’-AGGCGGACATTCTGGAAATG-3′
HES1
R：5’-CGGTACTTCCCCAGCACACTT-3′
F：5’- GATGTCCACACTGCCCAAGT-3’
MYBL2
R：5’-CAGGTGTCGTGAAGTGGCTT-3
F：5’- GGATCACCTCAACAGTCACGTC-3’
MAZ
R：5’-GGCACTTTCTCCTCGTGTCGTA-3’
F：5'- GACAACTTTGGCATCGTGGA-3'
GAPDH
R：5'-ATGCAGGGATGATGTTCTGG-3'
Supplementary figure legends
Supplementary Fig 1. Mutation types and violin plots of mutated genes.
Violin plots depicting the expression levels of the mutated genes in the 25
clusters, including PTEN (p.C71fs*3; D24fs*20), KMT2D (p.G2493V), PDGFRA
(p.T281M), STK11 (p.354L), and CTNNA1 (p.D265E) mutations in primary
samples (a) and KDM6A (p. Q191X) and TP53 (c.920-5C>T) mutations in lung
metastases (b). Some mutated genes showed aberrant expression in specific
cancer cell clusters.
Supplementary Fig 2. Distribution of different clusters in six samples and
their uniquely enriched genes. (a) Frequency distribution plots and their
values in six samples. (b) Heatmap of DEGs showing enriched genes for each
cell cluster.
Supplementary Fig 3. Definition of cell types based on unique genes. (a)
The definitions of the cell types are shown by a t-SNE plot and known marker
genes. (b) t-SNE plots showing the expression of known marker genes of each
cell cluster. The blue dashed boxes highlight the unique genes and known stem
cell genes in cluster 10.
Supplementary Fig 4. Analysis of the number and GO function of cluster
10. (a) Bubble graph showing the number of cell clusters (size scale) and the
z-scores (colour scale) for the DEGs. (b) GO analysis of 4 cell subtypes after
sub-clustering of cluster 10.
Supplementary Fig 5. Analysis of the unique genes and trajectories of
subcluster 10. (a) t-SNE plots showing the expression of unique marker genes
of subtype 3 from cluster 10. (b) Pseudotime trajectory analysis of all subtype
cells from cluster 10 and all samples by Monocle 2. Trajectories of subtype 3
and the primary tumour (A) are highlighted in red dashed boxes.
Supplementary Fig 6. Identification of differentially expressed genes and
the interaction network for cell subtypes in cluster 10. (a) Heatmap
showing the top 50 DEGs in the four subtypes. (b) Top 20 DEGs. (c) The PPI
network established the interactions between 20 DEGs and MYB or NOTCH1.
Supplementary Fig 7. Circos plots of fusions in SACC-83 cells and SACC-
LM cells.
Supplementary Fig 8. Both NOTCH1 and MYB promote SACC cell growth
and lung metastasis. (a) Statistical analysis of cell proliferation, invasion, and
colony formation at different time points after NOTCH1 knockdown in SACC-83
cells or MYB knockdown in SACC-LM cells. The p values were calculated using
the paired two-tailed Student’s t-test. Triple repeat data are presented as the
mean±SEM. p<0.05 (*), p<0.01 (**), p<0.001 (***). (b) HE images showing lung
metastases induced by the left intraventricular injection of SACC-83-NOTCH1-
KD, SACC-LM-MYB-KD, or control cells at 28 days (4-5 per group). The
number of metastases was calculated and compared between groups (Fig. 3f).
Supplementary Fig 9. The reciprocal interactions between NICD1 and
MYB or between MYC and MYB predicted with AlphaFold. Crystal
structures of NICD1 (1754-2555) and MYC (350-439) were obtained from NCBI
Structure (PDB ID: 6PY8; PDB ID: 5I4Z). The crystal structures of MYB (200-
500) and MYC (100-300) were predicted with AlphaFold2. Interactive docking
of protein‒protein complexes was performed in the ZDOCK server. Images
were exported from PyMOL 2.5.
