Supplementary Figures

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

non del del

- -

NOTCH NOTCH - del

NOTCH

mut mut

vs. vs. NOTCH

mut

non

vs. NOTCH

- del

WT - NOTCH

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 Supplementary Fig. S5

Cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT)

100 del-NOTCHmut (88) non-del-NOTCHmut (76) NOTCHWT (717) *

) 10

(

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 est iva cy s s s est va est iva op op lls me ma lls 4 re cti r h (T a r ct no ge ge ge r cti r ct in utr 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 e 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 non-del vs. WT 1.39 <0.001 r

r 0.3 non-del vs. WT 1.33 0.100 0.3

t 0.2 0.2

e m

m 0.1 0.1

r n

n 0.0 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 r

r 0.3 non-del vs. WT 2.04 <0.001 0.3 non-del vs. WT 1.71 <0.001

c s

s 0.2

t 0.2

n e

e 0.1 m

m 0.1 h

h 0.0

i r

r -0.1 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 r

0.3 non-del vs. WT 0.88 0.765 r 0.3 non-del vs. WT 1.30 0.111

0.2 t 0.2

e m

0.1 m 0.1

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

t 0.3

t 0.1

e m

0.2 m 0.0

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. naive CD8 T cell (up) Activated vs. naive CD8 T cell (down)

0.5 0.3 Comparison NES P value Comparison NES P value

0.4 del vs. WT 1.80 <0.001 0.2 del vs. WT -1.60 <0.001

del vs. non-del 1.51 0.012 del vs. non-del -1.32 0.063

e e r

r 0.3 non-del vs. WT -0.89 0.756 0.1 non-del vs. WT -0.85 0.826

o o

c c

s s

t 0.2 0.0

n n

e e m

m 0.1 -0.1

h h

c c

i i

r r n

n 0.0 -0.2

E E

-0.1 -0.3

-0.2 -0.4 0 5000 10000 15000 20000 0 5000 10000 15000 20000 Rank in ordered dataset Rank in ordered dataset

E Immunology F Immunology Induced vs. natural Treg (up) Induced vs. natural Treg (down)

0.4 0.6 Comparison NES P value Comparison NES P value del vs. WT 2.63 <0.001 0.3 del vs. WT -1.29 <0.001 0.5

del vs. non-del -1.20 0.100 del vs. non-del 1.46 <0.001

e e

r 0.4

r 0.2 non-del vs. WT -0.93 0.593 non-del vs. WT 1.83 <0.001

t 0.3

t 0.1

e m

m 0.0 0.2

r n

n -0.1 0.1

E E

-0.2 0.0

-0.3 -0.1 0 5000 10000 15000 20000 0 5000 10000 15000 20000 Rank in ordered dataset Rank in ordered dataset

G Immunology H Immunology Activated vs. naive NKT cell (up) Activated vs. naive NKT cell (down)

0.5 Comparison NES P value 0.3 Comparison NES P value del vs. WT 1.39 <0.001 del vs. WT -0.92 0.595 0.4 0.2

del vs. non-del -0.84 1.000 del vs. non-del 1.01 0.400

e r

0.3 non-del vs. WT 1.66 <0.001 r 0.1 non-del vs. WT -1.29 0.064

0.2 t 0.0

e m

0.1 m -0.1

r n

0.0 n -0.2

E E

-0.1 -0.3

-0.2 -0.4 0 5000 10000 15000 20000 0 5000 10000 15000 20000 Rank in ordered dataset Rank in ordered dataset Supplementary Fig. S9

A Reactome B Reactome Programmed cell death Metabolism of steroid hormones

0.5 Comparison NES P value 0.5 Comparison NES P value 0.4 0.4 del vs. WT 1.45 <0.001 del vs. WT 1.28 0.053

del vs. non-del 1.17 0.181 0.3 del vs. non-del 1.16 0.233

e e

r 0.3

non-del vs. WT 0.91 0.625 r non-del vs. WT -1.25 0.191 o

o 0.2

t 0.2

t 0.1

e m

0.1 m 0.0

i r

r -0.1 n

0.0 n E E -0.2 -0.1 -0.3

-0.2 -0.4 0 5000 10000 15000 20000 0 5000 10000 15000 20000 Rank in ordered dataset Rank in ordered dataset Supplemental Figure S10

A B NOTCH1 POPLAR/OAK cohort (atezolizumab) - PFS NOTCH2 POPLAR/OAK cohort (atezolizumab) - PFS

100 ORR (%) mPFS (mo) 100 ORR (%) mPFS (mo)

% mis-high+trunc % mis-high+trunc

l NOTCH1 (18) 27.8 8.92 l NOTCH2 (9) 18.2 5.75

a a

v mis-low v mis-low i NOTCH1 (5) 20.0 2.86 i NOTCH2 (5 ） 20.0 2.86 v 75 v 75 r WT r WT

u NOTCH (310) 14.5 2.56 u NOTCH (310) 14.5 2.56

s s

e e

r r

f f

- 50 - 50

n n

o o

i i

s s

e e

r 25 r 25

g g

o o

r r

P P 0 0 0 5 10 15 20 25 0 5 10 15 20 25 Time (months) Time (months) C D NOTCH3 POPLAR/OAK cohort (atezolizumab) - PFS NOTCH4 POPLAR/OAK cohort (atezolizumab) - PFS

100 ORR (%) mPFS (mo) 100 ORR (%) mPFS (mo)

% mis-high+trunc % mis-high+trunc

l NOTCH3 (6) 50.0 7.21 l NOTCH4 (9) 0.0 5.52

a a

v mis-low v mis-low i NOTCH3 (6) 0.0 3.48 i NOTCH4 (6) 0.0 1.41 v 75 v 75 r WT r WT

u NOTCH (310) 14.5 2.56 u NOTCH (310) 14.5 2.56

s s

e e

r r

f f

- 50 - 50

n n

o o

i i

s s

e e

r 25 r 25

g g

o o

r r

P P 0 0 0 5 10 15 20 25 0 5 10 15 20 25 Time (months) Time (months) Supplementary Fig. S11

A TCGA cohort (LUAD) - DFS B TCGA cohort (LUAD) - OS

100 mDFS (mo) 100 mOS (mo)

del-NOTCHmut (17) 67.2 del-NOTCHmut (36) 56.7

l mut mut

a non-del-NOTCH (29) 100.1 n.s. non-del-NOTCH (46) 52.6 n.s.

% v

75 75

l v

WT a WT

r NOTCH (208) 97.0 NOTCH (347) 50.2

s e

50 50

e O

s 25 25

i D

0 0 0 50 100 150 200 250 0 50 100 150 200 250 Time (months) Time (months) C D TCGA cohort (LUSC) - DFS TCGA cohort (LUSC) - OS

100 100 mOS (mo)

del-NOTCHmut (52) 54.4

l mut

a non-del-NOTCH (31) 97.9 n.s.

% v

75 75

l v

a WT

r NOTCH (362) 54.4

s e

50 50

l l

f mDFS (mo)

a r

e mut s

del-NOTCH (39) N.A. e

a e

mut O s 25 n.s. 25

i non-del-NOTCH (21) N.A. D NOTCHWT (223) N.A.

0 0 0 50 100 150 200 250 0 50 100 150 200 250 Time (months) Time (months) Supplementary Fig. S12

NSCLC (ITT population)

With EGFR mutation or ALK translocation

EGFR/ALKWT NSCLC

Without any NOTCH mutation

EGFR/ALKWT NSCLC with NOTCH1-4 mutation

Only with NOTCH4 mutation

EGFR/ALKWT NSCLC with NOTCH1/2/3 mutation

Only with NOTCH1/2/3mis-low mutation

EGFR/ALKWT NSCLC with EGFR/ALKWT NSCLC with EGFR/ALKWT NSCLC NOTCH1/2/3mis-high+trunc mutation NOTCH1/2/3mis-low or NOTCH4mutation without NOTCH mutation

Deleterious NOTCH mutation Non-deleterious NOTCH mutation NOTCHWT

Investigation of molecular mechanism

Higher TMB Higher TMB Lower TMB Higher DDR signature Lower DDR signature Lower DDR signature Higher infiltration of M1 Mφ Lower infiltration of M1 Mφ Lower infiltration of M1 Mφ Higher immune signature Lower immune signature Lower immune signature

Clinical benefit in RCTs: atezolizumab vs. docetaxel (POPLAR/OAK)

Strong Moderate Limited ORR: 29.4% vs. 5.6% ORR: 10.7% vs. 10.3% ORR: 14.5% vs. 13.3% PFS HR: 0.45 PFS HR: 0.65 PFS HR: 0.91 OS HR: 0.48 OS HR: 0.51 OS HR: 0.67

Deleterious NOTCH mutation in other cohorts

Immunotherapeutic cohorts Non-immunotherapeutic cohorts 1. Rizvi-PFS: multivariable HR=0.56, p=0.047 TCGA cohort: deleterious NOTCH mutation is not 2. MSKCC-OS: multivariable HR=0.54, p=0.049 associated with both DFS and OS