Supplementary Fig. S1 NOTCH1 NOTCH2 NOTCH3 NOTCH4 Co-mutation POPLAR/OAK (n=255) Rizvi/MSKCC (n=49) LUSC TCGA (n=469) Total (n=773) POPLAR/OAK (n=598) Rizvi/MSKCC (n=299) Non-LUSC TCGA (n=562) Total (n=1159) 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 Mutational rate (%) Mutational rate (%) Mutational rate (%) Mutational rate (%) Mutational rate (%) Supplementary Fig. S2 A POPLAR/OAK Univariable analysis Multivariable analysis PFS HR (95% CI) p value HR (95% CI) p value NOTCH1 (mut, WT) 0.70 (0.43-1.13) 0.140 0.69 (0.43-1.10) 0.121 NOTCH2 (mut, WT) 0.89 (0.52-1.53) 0.680 0.87 (0.51-1.48) 0.601 NOTCH3 (mut, WT) 0.74 (0.40-1.39) 0.354 0.73 (0.39-1.36) 0.320 NOTCH4 (mut, WT) 1.21 (0.74-1.98) 0.442 1.23 (0.75-2.00) 0.415 NOTCH1/2/3 (mut, WT) 0.72 (0.52-1.00) 0.051 0.25 0.5 1 2 4 0.25 0.5 1 2 4 Favors NOTCHmut Favors NOTCHWT Favors NOTCHmut Favors NOTCHWT B Rizvi Univariable analysis Multivariable analysis PFS HR (95% CI) p value HR (95% CI) p value NOTCH1 (mut, WT) 0.69 (0.34-1.41) 0.313 0.65 (0.32-1.34) 0.244 NOTCH2 (mut, WT) 0.47 (0.17-1.27) 0.136 0.41 (0.14-1.18) 0.098 NOTCH3 (mut, WT) 0.67 (0.30-1.51) 0.329 0.63 (0.28-1.42) 0.266 NOTCH4 (mut, WT) 0.92 (0.47-1.80) 0.799 1.23 (0.60-2.52) 0.570 NOTCH1/2/3 (mut, WT) 0.56 (0.34-0.92) 0.023 0.25 0.5 1 2 4 0.25 0.5 1 2 4 Favors NOTCHmut Favors NOTCHWT Favors NOTCHmut Favors NOTCHWT C POPLAR/OAK Univariable analysis Multivariable analysis OS HR (95% CI) p value HR (95% CI) p value NOTCH1 (mut, WT) 1.01 (0.60-1.71) 0.963 1.00 (0.59-1.68) 0.987 NOTCH2 (mut, WT) 0.94 (0.50-1.77) 0.847 0.91 (0.48-1.72) 0.767 NOTCH3 (mut, WT) 0.68 (0.32-1.45) 0.317 0.69 (0.32-1.46) 0.329 NOTCH4 (mut, WT) 1.30 (0.76-2.22) 0.347 1.29 (0.75-2.21) 0.362 NOTCH1/2/3 (mut, WT) 0.85 (0.58-1.24) 0.393 0.25 0.5 1 2 4 0.25 0.5 1 2 4 Favors NOTCHmut Favors NOTCHWT Favors NOTCHmut Favors NOTCHWT D MSKCC Univariable analysis Multivariable analysis OS HR (95% CI) p value HR (95% CI) p value NOTCH1 (mut, WT) 0.43 (0.18-1.05) 0.064 0.44 (0.18-1.07) 0.070 NOTCH2 (mut, WT) 0.50 (0.19-1.36) 0.175 0.51 (0.19-1.40) 0.192 NOTCH3 (mut, WT) 0.65 (0.27-1.58) 0.340 0.61 (0.25-1.49) 0.277 NOTCH4 (mut, WT) 0.98 (0.54-1.81) 0.959 1.11 (0.60-2.06) 0.748 NOTCH1/2/3 (mut, WT) 0.48 (0.27-0.84) 0.010 0.25 0.5 1 2 4 0.25 0.5 1 2 4 Favors NOTCHmut Favors NOTCHWT Favors NOTCHmut Favors NOTCHWT C B A Supplementary Fig Supplementary non del del - - NOTCH NOTCH - del - NOTCH mut mut vs. NOTCH vs. mut non vs. NOTCH vs. S3 - del WT - NOTCH WT mut Supplementary Fig. S4 group RFC4 USP1 p = 0.0136 ATR UBE2I p = 0.0274 TOPBP1 TOPBP1 p = 0.0105 ISY1 SUMO3 p = 0.0390 MBD4 RPA1 p = 0.0935 RFC4 POLR2K p = 0.0623 RFC1 p = 0.0686 GTF2H4 RBBP8 p = 0.0741 FANCE RAD51AP1 p = 0.0979 PNKP POLR2K p = 0.0518 LIG1 POLD3 p = 0.0876 UBE2I PNKP p = 0.0434 PALB2 PALB2 p = 0.0411 COPS8 Z scoreNFRKBgroup p = 0.0979 4 group MAD2L2 MSH6 wt p = 0.0945 2 mut wt SUMO3 MSH2 p = 0.0160 0 mut EME1 -2MBD4 p = 0.0149 RAD51AP1 MAD2L2-4 p = 0.0231 CHEK1 LIG1 p = 0.0289 FANCB ISY1 p = 0.0143 CDK1 GTF2H4 p = 0.0715 FANCL FANCL p = 0.0758 POLD3 FANCE p = 0.0256 FANCC RBBP8 p = 0.0761 FANCB p = 0.0833 MSH6 EME1 p = 0.0954 MSH2 COPS8 p = 0.0420 USP1 CHEK1 p = 0.0086 FANCC CDK1 p = 0.0691 RPA1 ATR p = 0.0167 NFRKB 0 5 10 15 20 25 RFC1 Z score Cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) 100 del-NOTCHmut (88) non-del-NOTCHmut (76) NOTCHWT (717) Supplementary* ) 10 % ( n o i Fig. t r o S5 p o r 1 P 0.1 r ) ive ory ells D8 ive ing ted pe gs lta ing ted tes M0 M1 M2 ing ted ing ted hils hils na m c C na st va el re de st va cy s s s st va st va op p lls me ma lls 4 re cti r h (T a re cti no ge ge ge re cti re cti in tro ce lls as ce CD ory y a ula ry mm ells s a Mo ha ha ha ells s a ells s a os eu B ce Pl T lls m or llic lato ga c ell rop rop rop c ell t c ell E N B ce me em fo gu lls NK c ac ac ac ritic c c as t c T 4 m lls re ce NK M M M nd riti M as CD 4 ce lls T e nd M lls CD T ce D De ce ells T T T c Supplementary Fig. S6 A Reactome B Reactome Antigen processing: ubiquitination degradation MHC-I mediated antigen presentation 0.5 Comparison NES P value 0.5 Comparison NES P value del vs. WT 1.08 0.270 0.4 del vs. WT 1.58 <0.001 0.4 del vs. non-del 1.05 0.325 del vs. non-del -0.90 0.786 e e r 0.3 non-del vs. WT 1.33 0.100 r 0.3 non-del vs. WT 1.39 <0.001 o o c c s s t 0.2 t 0.2 n n e e m 0.1 m 0.1 h h c c i i r r n 0.0 n 0.0 E E -0.1 -0.1 -0.2 -0.2 0 5000 10000 15000 20000 0 5000 10000 15000 20000 Rank in ordered dataset Rank in ordered dataset C Reactome D Reactome MHC-II mediated antigen presentation Antigen processing: cross presentation 0.6 Comparison NES P value 0.5 Comparison NES P value 0.5 del vs. WT -1.13 0.233 del vs. WT 1.38 0.029 0.4 0.4 del vs. non-del -1.92 <0.001 del vs. non-del -0.94 0.692 e e r 0.3 non-del vs. WT 2.04 <0.001 r 0.3 non-del vs. WT 1.71 <0.001 o o c c s 0.2 s t t 0.2 n n e 0.1 e m m 0.1 h 0.0 h c c i i r -0.1 r n n 0.0 E E -0.2 -0.1 -0.3 -0.4 -0.2 0 5000 10000 15000 20000 0 5000 10000 15000 20000 Rank in ordered dataset Rank in ordered dataset Supplementary Fig. S7 A Reactome B Reactome BCR downstream signaling TCR downstream signaling 0.5 0.5 Comparison NES P value Comparison NES P value 0.4 del vs. WT 1.37 0.018 0.4 del vs. WT 1.54 0.014 del vs. non-del 0.97 0.500 del vs. non-del 0.81 0.931 e e r 0.3 non-del vs. WT 0.88 0.765 r 0.3 non-del vs. WT 1.30 0.111 o o c c s s t 0.2 t 0.2 n n e e m 0.1 m 0.1 h h c c i i r r n 0.0 n 0.0 E E -0.1 -0.1 -0.2 -0.2 0 5000 10000 15000 20000 0 5000 10000 15000 20000 Rank in ordered dataset Rank in ordered dataset Supplementary Fig. S8 A Immunology B Immunology Activated vs. naive CD4 T cell (up) Activated vs. naive CD4 T cell (down) 0.6 Comparison NES P value 0.4 Comparison NES P value del vs. WT 2.22 <0.001 del vs. WT -1.24 0.094 0.5 0.3 del vs. non-del 1.50 0.012 del vs. non-del -1.10 0.118 e e r 0.4 non-del vs. WT 1.45 <0.001 r 0.2 non-del vs. WT -1.05 0.358 o o c c s s t 0.3 t 0.1 n n e e m 0.2 m 0.0 h h c c i i r r n 0.1 n -0.1 E E 0.0 -0.2 -0.1 -0.3 0 5000 10000 15000 20000 0 5000 10000 15000 20000 Rank in ordered dataset Rank in ordered dataset C Immunology D Immunology Activated vs.