Supplementary Table S1. ATM alterations in the 1,661-patient MSK-TMB study

Cancer Type Total Mutation Fusion Bladder Cancer 215 23 0 Colorectal Cancer 110 11 0 Melanoma 320 26 0 Cancer of Unknown Primary 88 7 0 Non-Small Cell Lung Cancer 350 23 0 Esophagogastric Carcinoma 126 6 1 Breast Cancer 44 2 0 Glioma 117 3 0 Renal Cell Carcinoma 151 2 0 Head and Neck Cancer 139 1 0

Supplementary Table S2. ATM alternations in the 10,945-patient MSK-IMPACT study

Cancer Type Total Mutation Deep deletion Amplification Multi-alterations Fusion Small Bowl Cancer 35 7 0 0 0 0

Skin Cancer, Non-Melaoma 148 19 1 0 0 0 Bladder Cancer 423 45 2 0 0 0

Endometrial Cancer 218 24 0 0 0 0

Hepatobiliary Cancer 355 27 0 0 0 0 Colorectal Cancer 1007 75 0 0 0 1 Mature B-Cell Neoplasms 134 8 0 0 2 0 Non-Small Cell Lung Cancer 1668 120 0 2 1 0 Melanoma 365 23 0 0 0 0 Appendiceal Cancer 79 4 0 0 0 0

Small Cell Lung Cancer 82 4 0 0 0 0

Prostate Cancer 717 25 7 1 0 0 Histiocytosis 22 1 0 0 0 0 Salivary Gland Cancer 114 5 0 0 0 0 Thyroid Cancer 231 10 0 0 0 0 Cancer of Unknown Primary 186 8 0 0 0 0 Breast Cancer 1324 48 3 2 0 0

Adrenocortical Carcinoma 25 1 0 0 0 0 Mature T and NK Neoplasms 29 1 0 0 0 0 Renal Cell Carcinoma 361 12 0 0 0 0 Head and Neck Cancer 186 14 0 0 0 1 Pancreatic Cancer 502 69 7 2 0 0 Glioma 553 14 1 0 0 0 Soft Tissue Sarcoma 443 13 1 0 0 0 Esophagogastric Cancer 341 8 1 0 0 0 Peripheral Nervous System 80 0 2 0 0 0 Germ Cell Tumor 288 2 0 1 0 1 Ovarian Can 244 2 1 0 0 0 Uterine Sarcoma 93 1 0 0 0 0 Mesothelioma 107 1 0 0 0 0

Bone Cancer 134 1 0 0 0 0 Gastrointestinal Stromal Ca 137 0 0 0 0 1 Supplementary Table S3. SgRNA used in CRISPR/Cas9-mediated knockouts

Target Sequence

Human-ATM-sg1 TTGTTTCAGGATCTCGAATC Human-ATM-sg2 GATGCAGGAAATCAGTAGTT Mouse-ATM-sg1 CTCTGTCATGCTCTAACTGC Mouse-ATM-sg2 CTGTTTCAGGATCCTGAATC Mouse-TFAM-sg1 ACGGGGGTCGAGATGTGCGC Mouse-TFAM-sg2 TACCAGCGTGGGAACTCCGG Mouse-cGAS CAGAATGCAGAAACGGGAGT Mouse-TBK TGCCGTTTAGACCCTTCGAG Mouse-Sting CTTCTCGCTACAACACATGA Mouse-MDA5 GGCAGGGATTCAGGCACCAT

Supplementary Table S4. Oligonucleotide primers for qPCR

Target Sequence

Mouse-β-actin (F) GAAATCGTGCGTGACATCAAA

Mouse-β-actin (R) TGTAGTTTCATGGATGCCACA

Mouse-IFNβ1 (F) CTGGCTTCCATCATGAACAA

Mouse-IFNβ1 (R) AGAGGGCTGTGGTGGAGAA

Mouse-IFNɑ (F) GGATGTGACCTTCCTCAGACTC

Mouse-IFNɑ (R) ACCTTCTCCTGCGGGAATCCAA

Mouse-CCL5 (F) CAAGTGCTCCAATCTTGCAGTC

Mouse-CCL5 (R) TTCTCTGGGTTGGCACACAC

Mouse-IFIT1 (F) CAAGGCAGGTTTCTGAGGAG

Mouse-IFIT1 (R) GACCTGGTCACCATCAGCAT

Mouse-ISG15 (F) CTAGAGCTAGAGCCTGCAG

Mouse-ISG15 (R) AGTTAGTCACGGACACCAG

Mouse-mtDNA Dloop1 (F) AATCTACCATCCTCCGTGAAACC

Mouse-mtDNA Dloop1 (R) TCAGTTTAGCTACCCCCAAGTTTAA

Mouse-mtDNA Dloop2 (F) CCCTTCCCCATTTGGTCT

Mouse-mtDNA Dloop2 (R) TGGTTTCACGGAGGATGG

Mouse-mtDNA Dloop3 (F) TCCTCCGTGAAACCAACAA

Mouse-mtDNA Dloop3 (R) AGCGAGAAGAGGGGCATT

Mouse-mtDNA CytB (F) GCTTTCCACTTCATCTTACCATTTA

Mouse-mtDNA CytB (R) TGTTGGGTTGTTTGATCCTG

Mouse-mtDNA 16S (F) CACTGCCTGCCCAGTGA

Mouse-mtDNA 16S (R) ATACCGCGGCCGTTAAA

Mouse-mtDNA ND1(F) CTAGCAGAAACAAACCGGGC

Mouse-mtDNA ND1 (R) CCGGCTGCGTATTCTACGTT

Mouse-mtDNA ND4 (F) AACGGATCCACAGCCGTA

Mouse-mtDNA ND4 (R) AGTCCTCGGGCCATGATT

Mouse-mtDNA-Cox1 (F) GCCCCAGATATAGCATTCCC

Mouse-mtDNA-Cox1 (R) GTTCATCCTGTTCCTGCTCC

Mouse-nucDNA HK2 (F) GCCAGCCTCTCCTGATTTTAGTGT Mouse-nucDNA HK2 (R) GGGAACACAAAAGACCTCTTCTGG

Mouse-nucDNA Tert (F) CTAGCTCATGTGTCAAGACCCTCTT

Mouse-nucDNA Tert (R) GCCAGCACGTTTCTCTCGTT

Mouse-nucDNA PTGER2 (F) CCTGCTGCTTATCGTGGCTG

Mouse-nucDNA PTGER2 (R) GCCAGGAGAATGAGGTGGTC

Mouse-nucDNA NDUFV1 (F) CTTCCCCACTGGCCTCAAG

Mouse-nucDNA NDUFV1 (R) CCAAAACCCAGTGATCCAGC A B Datans 60 202000 4T1 VC 151500

ɑ-ATM 101000 Colony ɑ−GAPDH ATM KO Colony# 5050

00 VC ATM KO NSG C NSG D

) 2000 ATM KO 3 4T1 in NSG 100 2000) 100

3 ATM KO VC VC 1500 1500 VC (n=5) VC (n=5) ATM KO (n=5) 10001000 ATM KO (n=5) ns 5050

500 500 survival Percent Percent Percent survival Tumor volume(mm Tumor 0 Tumor volume(mm Tumor 0 0 0 5 10 15 0 0 5 10 15 05 10 1520 25 DaysDays postpost-tumor-tumor injection injection Days post-tumorTime injection G E F ɑPD1 Ab(100µg,i.p.) ɑPD1 Ab(100µg,i.p.) B16

ɑ-ATM 0 6 9 12 0 6 9 12 AZD1390 (D4-D14, Daily injection) ɑ−GAPDH 100K tumor cells injected 100K tumor cells injected

Supplementary Figure 1. ATM knockout and its effect on tumor growth in vitro and in vivo. (A) Western blot analysis of ATM expression in control and ATMKO 4T1 tumor cells . (B) Clonogenic abilities of vector control and ATM KO 4T1 cells. Cell were seeded in 6-well plates at 500 cells per well in triplicates and allowed to grow for 6 days before staining with crystal violet (left panel). The colony number were counted by use of Image J and plotted (right panel). (C-D) Tumor volume (C) and Kaplan-Meier survival curve (D) of immunodeficient NSG mice inoculated with about 1x105 control or ATMKO 4T1 cells. (E) Western Blot analysis of ATM expression of vector control and ATMKO murine B16F10 tumor cells. (F) Treatment schedule for tumor growth delay experiments in Fig.1E, 1F. (G) Treatment schedule of tumor growth delay experiments in Fig.1G, 1H, 1I, 1J. Error bars represent ±SEM. NS, not significant, as determined by unpaired Student's t test (B and C) or log-rank test (D). A B

Enrichment plot: GO_IMMUNE_RESPONSE Enrichment plot: GO_INNATE_IMMUNE_RESPONSE 0.05 0.00 0.00 -0.05 -0.05 -0.10 -0.10 -0.15 -0.15 CFP -0.20 ASS1 -0.20 TMEM173 -0.25 ISG15 -0.25 IFIH1 -0.30 NES = -2.137168 -0.30 NES = -1.9525625 IFIT1 -0.35 FOXJ1 (ES) Enrichmentscore -0.35 -0.40 CSF2 -0.40 CCL5

Enrichment score (ES) Enrichmentscore p = 0 -0.45 p = 0 SLPI -0.45 ASS1 -0.50 ULBP1 ISG15 NCF4 SLPI SPN ULBP1 GBP2 2 GBP2 2 TNFRSF14 1 1 0 0 -1 -1 -2 -2 -3 -3

0 1000 2000 3000 4000 5000 6000 7000 8000 9000 metric(Signal2Notes)Rankedlist 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 Ranked list metric(Signal2Notes)Rankedlist

C Enrichment plot: GO_POSITIVE_REGULATION_OF_CYTOKINE_SECRETION

0.0 -0.1 -0.2 -0.3 -0.4 -0.5 NES = -2.35107 -0.6 Enrichment score (ES) Enrichmentscore -0.7 p = 0

2 1 0 -1 -2 HYAL2 -3 CLEC5A 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 HMGB1 Ranked list metric(Signal2Notes)Rankedlist AIM2 PTGER4 PANK1 CSF1R LGALS9 RLTPR TNFRSF14

Supplementary Figure 2. Upregulation of expression of involved in innate immune response and cytokine secretion in ATM-deficient 4T1 cells in vitro. Gene set enrichment analysis (GSEA) of genes in involved in immune response (A), innate immune response (B), and cytokine secretion (C) in vector control and ATMKO 4T1 cells. FDR calculated using GSEA. A B16F10 B HFF Dox(µg/ml) 0 1 5 ɑ-cGAS ɑ-p-TBK ɑ -p-TBK ɑ-TBK ɑ-TBK ɑ-Sting

ɑ-ATM ɑ-ATM

ɑ-actin ɑ-GAPDH

C D MDA-MB-231 HFF 0 0.625 1.25 2.5 5 AZD (µM) 0 0.625 1.25 AZD(µM) ɑ-p-ATM ɑ-p-TBK ɑ-ATM ɑ-TBK ɑ -p-TBK ɑ-p-ATM ɑ-TBK ɑ-ATM

ɑ-actin ɑ-GAPDH

E F B16F10 MDA-MB-231 Ku (µM) 0 0.625 1.25 2.5 5 10 Ku (µM) 0 0.625 1.25 2.5 5 10 ɑ-p-TBK ɑ-p-TBK ɑ-TBK ɑ-TBK ɑ-ATM ɑ-GAPDH ɑ-GAPDH B16F10 G H b16Ku Control Ku Ku(h) 0 1 2 3 6 9 1010 **** ɑ-p-TBK 88 **** ɑ-TBK 66 ɑ-ATM 44 22 ɑ-GAPDH 0

Relative expression(fold) Relative 0 IFIifit1T1 CCccl5L5

Supplementary Figure 3. Additional data on ATM inhibition induced cGAS-STING activation in malignant and normal cells. (A ) WB analysis of p-TBK, TBK, cGas and ATM in B16F10 cells that had been transfected with inducible ATM shRNA and treated with doxycycline at indicated concentrations for 4 days. (B) WB analysis of p-TBK, TBK, STING and ATM in vector control(VC) and ATM KO human forskin fibroblast (HFF) cells. (C) WB analysis of p-ATM and p-TBK levels in MDA-MB- 231 cells treated with AZD1390 at indicated concentrations for 48 hrs. (D) WB analysis of p-ATM and p-TBK levels in HFF cells treated with AZD1390 at indicated concentrations for 48 hrs. . (E) WB analysis of p-TBK and TBK in MDA-MB-231 cells treated with Ku55933 at indicated concentrations for 6 hrs. (F) WB analysis of p-TBK and TBK in B16F10 cells treated with 5 µM Ku55933 at indicated times. (G) WB analysis of p-TBK and TBK in B16F10 cells treated with Ku55933 (10 µM) for different times. (H) Transcription levels of IFIT1 and CCL5 in B16F10 cells treated with 10µM Ku55933 for 9 hrs and analyzed by real- time PCR. Error bars represent SEM, n=3, ***p<0.001, as determined by 2-way ANOVA lich A LIHClich LIHC LIHClich 1313 1313 1818 r = -0.3193 p < 0.0001 11 9 11 12 IRF3

IRF7 9 12 ISG15 r = -0.1683 r = -0.3778 IRF3 (Log2) IRF3 IRF7 (Log2) IRF7 p = 0.0011 9 p < 0.0001 (Log2) ISG15 55 9 66 6 8 10 12 6 8 10 12 6 8 10 12 6 8 ATM 10 12 6 8 ATM 10 12 6 8 10 12 ATM(Log2) ATM(Log2) ATM(Log2)ATM prad B prad prad PRAD PRAD PRAD 16 1212 1212 16 1111 10 10 11 10 11 (Log2)

10IRF3 IRF7 99 ISG15 r = -0.498 8 r = -0.473 r = -0.3141 IRF7

IRF3 (Log2) IRF3 8 ISG15 (Log2) ISG15 p < 0.0001 7 p < 0.0001 66 p < 0.0001 8 7 8 7 8 9 10 11 12 7 8 9 10 11 12 7 8 9 10 11 12 r =7 - 0.26378 9 10 11 12 7 8 9 10 11 12 7 8 9 10 11 12 ATM r = - 0.1987 ATM ATM(Log2)ATM p < 0.0001ATM(Log2) p < 0.0001 ATM(Log2) COAD r = - 0.3044 COAD COAD p < 0.0001 COAD C 1212 COAD COAD 1212 1414 1111 1010 10 IRF3 10 10 IRF7 10 8 ISG15 IRF3 (Log2) IRF3 r = -0.2637 8 IRF7 (Log2) IRF7 r = -0.1987 r = -0.3044 99 p < 0.0001 66 p < 0.0001 (Log2) ISG15 66 p < 0.0001 6 7 8 9 10 11 6 7 8 9 10 11 6 7 8 9 10 11 6 7 8 9 10 11 6 8 10 12 ATM(Log2)ATM ATM 6 8 10 12 ATM (Log2) ATM(Log2)ATM

Supplementary Figure 4. Correlation between ATM and ISG gene transcriptional levels. Correlation analysis for ATM expression level versus IRF3, IRF7, and ISG15 in human (A) liver hepatocellular carcinoma (LIHC, 366 samples), (B) prostate adenocarcinoma (PRAD, 498 samples), (C) colorectal adenocarcinoma (COAD, 437 samples) from TCGA Pan Cancer Atlas. R and p represent Pearson correlation coefficients and p values from two-tailed Student’s t-test. 4t1-m4T1ito 7.5 7.5 **** A B 6.06.0 VC ATM KO 4.54.5 ******** WCE Pel Cyt **** 3.03.0 *** *** ns ns ɑ-HSP60 1.51.5

ɑ (fold) DNA Cytosolic -GAPDH 0.00 ɑ rt 1 2 3 4 S B 1 -ATM tert p p p ND4D 16S6 t x e o o o 1 Cytby Cox1o -T Dloop1o Dloop2o Dloop3o -N - C C c l l l t t - t- u D D D m t N t- t- t- m m m m m m C DAPI Anti-DNA HSP60 VC ATM KO ATM

Supplementary Figure 5. Additional data validating mitochondrial DNA release as the key factor in ATM deficiency-mediated cGAS-STING activation. (A) WB analysis validating our cellular fractionation protocol. Vector control and ATM KO 4T1 cells were subjected to digitonin fractionation as described in the Methods. extracts (WCE), pellets (Pel) and cytosolic extracts(Cyt) were blotted using indicated antibodies. (B) DNA was extracted from digitonin extracts of vector control and ATM 4T1 cells. Cytosolic mtDNA was quantitated via qPCR using mitochondrial DNA primer sets and the TERT primer. Normalization was carried out as described in the Materials and Methods section. (C) Immunofluorescence detection of dsDNA location in vector control (VC) and ATM KO 4T1 cells by use of anti-dsDNA (green), anti-HSP60(red, for mitochondria), and DAPI (for nuclear DNA). Scale bar represents 10 µm. Error bars in B represent SEM, n=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant, as determined by 2-way ANOVA. 4T1 cells Supplementary Figure 6. Down A and mitotochondrial Enrichment plot: MITOCHONDRIAL_MATRIX

in in vitro Enrichment score (ES) 0.2 0.3 0.4 0.5 Ranked list metric(Signal2Notes)0.0 0.1 - - - 1 2 0 1 2 3 0 1000 p = 0 NES = 2.4905837 2000 . Geneset enrichmentanalysis(GSEA) of proteincomplexgenes (C) 3000 4000 5000 6000 C - 7000 regulation of expressionof genes involved inmitochondrial functions in ATM 8000 GO_MITOCHONDRIAL_PROTEIN_COMPLEX 9000

Enrichment0.15 score (ES) 0.00 0.10 0.05 0.20 0.25 0.30 0.40 0.35 Ranked list metric(Signal2Notes) 0.45 - - - 1 2 1 2 3 0 0 1000 in controland p = 0 = p 1.9367038 = NES 2000 Enrichment plot: 3000 ME2 FDXR AGXT2L2 MRPL48 ACAD8 DBT MTG1 SUCLG2 MIPEP MTHFD1L 4000 5000 mitochondrialmatrix genes(A), mitochondrialtranslation genes(B), ATM KO 4 B 6000 7000 8000 Enrichment plot: GO_MITOCHONDRIAL_TRANSLATION T1 9000

tumor Enrichment score (ES) 0.2 0.3 0.4 0.0 0.1 Ranked list metric(Signal2Notes) 0.5 - - - 1 2 0 1 2 3 0 1000 cells p = 0 NES = 2.155055 2000 3000 . DBT NDUFV1 COX8A NDUFB3 NDUFS7 ATP5G1 CHCHD6 NDUFA9 NDUFB8 NDUFSS 4000 5000 6000 7000 8000 9000 - deficient MRPL48 MRPL45 MTRF1 MRPS24 MRPL9 DAP3 MRPS30 DARS2 MRPS33 MTRF1 ddC A B ɑ-cGas **** **** 2020 VC ɑ-p-TBK ATM KO 1515 ATM KO +ddc ɑ-TBK 1010 *** ** ɑ-Sting 55

ɑ-ATM expression(fold) Relative 00 IFIifit1T1 CCccl5L5 ɑ-GAPDH

C Anti-dsDNA HSP60 DAPI Merge VC ATM KO ATM VC dd C ATM KO ATM

Supplementary Figure 7. Additional data validating cytoplasmic release of mtDNA being responsible for ATM inhibition induced cGAS-STING activation. (A) WB analysis of levels of pTBK, TBK, cGas and STING in control (VC) and ATM KO B16F10 cells exposed to 100 nM /ml ddC for 20 days to deplete mtDNA. (B) Q-PCR analysis of type I interferon response in vector control and ATMKO B16F10 cells that had been treated with 100 nM /ml ddC for 20 days to deplete mtDNA. (C) Control (VC) and ATM KO B16F10 cells that had been treated with 100 nM /ml ddC for 20 days were co-stained with anti-dsDNA (green), anti-HSP60(red) and DAPI. Scale bar indicated 10 µm. Error bars represent SEM, n=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant, as determined by 2-way ANOVA. A B HSP60 Anti-dsDNA DAPI Merge

** 1.21.2 *** VC

0.80.8 copy # (fold)# copy 0.40.4 mtDNA 0.00.0 vc tfko ako TFAM KO TFAM

TF KO-mito C D VC 2.5 TFAM KO WCE Pel Cyt **** **** **** **** **** 2.0 **** **** ns ɑ-GAPDH 1.5 ns

1.0 ɑ-Histon-H3

Cytosolic DNA (fold) CytosolicDNA 0.5 ɑ-HSP60 0.0 ɑ-TFAM t r 2 4 tB S 1 1 2 3 e K D y 6 x p p p ert2-T H N 16S-1 o o o o T HK2- ND4- CytB-C t C lo lo lo c c t t CoX1t- u u m m Dloop1D Dloop2D Dloop3-D N N m m t- t- t m m m

Supplementary Figure 8. Additional data validating mitochondrial DNA release as being involved in TFAM deficiency-mediated cGAS-STING activation. (A) qPCR analysis of mitochondrial copy number based on mitochondrial gene ND1 in vector control (VC) and TFAM KO or ATM KO B16F10 cells. (B) Immunofluorescence detection of dsDNA location in vector control (VC) and TFAM KO B16F10 cells by use of anti-dsDNA (green), anti-HSP60(red, for mitochondria), and DAPI (for nuclear DNA). Scale bar represents 10 µm. (C) WB analysis validating our cellular fractionation protocol. Vector control and TFAM KO B16F10 cells were subjected to digitonin fractionation as described in the Material and Methods section. Whole cell extracts (WCE), cell pellets (Pel) and cytosolic extracts(Cyt) were then blotted using indicated antibodies. (D) Cytosolic DNA was extracted from digitonin extracts of vector control and TFAM KO B16F10 cells. Cytosolic mtDNA was quantitated by qPCR using mitochondrial DNA primer sets and while nuclear DNA by use of the TERT gene primers. Normalization was carried out as described in the Materials and Methods section. Error bars in A & D represent SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 ns, not significant, as determined by 2-way ANOVA. A B WCE Pel Cyt Mito

VC+ EV ATM KO +EV ATM KO + HA-TFAM ɑ 88 -GAPDH *** *** Legend ****** Legendɑ 66 -Histon-H3 Legend 44 ɑ-HSP60

22 ɑ-HA-TFAM

Relative expression(fold) Relative 00 IFIIFIT1T1 CCCCL5L5 ɑ-ATM

C VC + EV HA-TF-mt ATM KO + EV 99 ATM KO + HA-TFAM **** vc **** ******** **** atm KO (fold) **** **** 66 atm KO + TF

mtDNA *** 33 Cytosolic Cytosolic

00 1 4 S B 1 2 3 D D X1 6 t p p p N N O 1 y o o o C C lo lo lo D D D

Supplementary Figure 9. Over-expression of TFAM in ATM KO cells inhibits release of mitochondria DNA and cGAS/STING activation. (A) Q-PCR analysis of interferon-stimulated gene expression in vector control and ATMKO B16F10 cells that had been transfected with empty vector (EV) or HA-TFAM. (B) WB verification of our cytosol fractionation protocol. Vector control and ATM KO B16 cells had been transfected with empty control (EV) or HA-TFAM were subjected to digitonin fractionation as described in the Methods and whole-cell extracts (WCE), pellets (Pel), cytosolic extracts(Cyt) and (Mito) were blotted using indicated antibodies. (C) Q-RT PCR quantification of cytosolic DNA extracted from digitonin- permeabilized cyotosolic extracts of control and ATM KO B16F10 cells that had been transfected with empty vector (EV) or HA- TFAM. Normalization was carried out as described in the Methods section. In A & C, error bars represent ±SEM, n=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 ns, not significant, as determined by two-way ANOVA. A VC ATM KO B VC + - - - - + - - - - cGas sgRNA - + - - - - + - - - TBK sgRNA - - + - - - - + - - VC Sting sgRNA - - - + - - - - + - 6060 *** *** *** ATM KO MDA5 sgRNA ATM/MDA5 DKO - - - - + - - - - + *** ɑ-cGas 4545

ɑ-TBK 3030 ɑ -Sting *** ns 1515 ɑ-MDA5 ɑ -ATM expression(fold) Relative 00 ifit1 ccl5 isg15 ɑ-GAPDH IFIT1 CCL5 ISG15

C VC (n=8) D MDA5 MDA5 sgRNA (n=5) ATM/MDA5 ATM sgRNA (n=8) 100100 sg Control (n=8) 2500 ns

2500) sg Control (n=8) ) ATM/MDA5 sgRNA (n=7) MDA5 KO (n=5) 3 3 20002000 MDA5 KO (n=5) ATM KO (n=8) ATM KO (n=8) ATM/MDA5 DKO (n=6) 15001500 50 ns ATM/MDA5 DKO50 (n=6) ns 10001000 ns survival Percent

500500 survival Percent 0

Tumor volume(mm Tumor 0 Tumor volume(mm Tumor 00 0 15 3030 4545 0 6 6 121218 1824 24 Days post-tumorDays injection Days post-tumorDays injection

ATM/Sting DKO + isotype

ATM/cGAS DKO + isotype ATM/Sting DKO + anti-PD1 E ATM/cGAS DKO + anti- F ATM/TBK DKO + isotype G PD1 ATM/TBK DKO + anti-PD1 ATM/cGAS DKO +isotype (n=5) ATM/TBK DKO +isotype (n=5) ATM/STING DKO +isotype (n=5) cGAS DKO TBK DKO ATM/cGAS DKO + anti-PD1 (n=5) ATM/TBK DKO + anti-PD1 (n=5) ATM/StingSting KO DKO+ anti-PD1 (n=5) 20002000 ) )

) 1500 3 ) 3 1500 2000 3 2000 ) ) ns 3 3 * ns 3 15001500 15001500 10001000 10001000 10001000 500 500 500 500 500500 Tumor volume(mm Tumor Tumor volume(mm Tumor Tumor volume(mm Tumor Tumor volume(mm Tumor Tumor volume(mm Tumor 00 volume(mm Tumor 0 00 0 10 20 30 10 20 30 0 1010 2020 3030 0 10 20 30 Days post-tumorDays injection Days postDays-tumor injection Days post-tumorDays injection

Supplementary Figure 10. MDA5 is not required for ATM deficiency mediates ISG activation and tumor growth delays. (A) WB verification of gene knockout for cGas, TBK, STING, and MDA5 in vector control (VC), and ATMKO cells. GAPDH was used as the protein loading control. (B) Transcriptional levels of interferon response genes in vector control (VC), ATM KO or or ATM/MDA5 DKO B16 cells analyzed by real-time qPCR. Error bars: SEM, n=3. ***p<0.001. (C-D) Tumor volume (C) and Kaplan-Meier survival curve (D) of C57BL/6 mice inoculated with 1x105 vector control (VC), MDA5 KO, ATM KO, or ATM/MDA5 DKO B16F10 cells. The control groups VC and ATMKO are the same as shown in Fig. 6D-I. (E-G) Tumor growth in C57BL/6 mice inoculated with 1x105 ATM KO or ATM/cGas DKO (E), ATM/TBK DKO (F), and ATM/STING DKO (G) B16F10 cells and treated with anti-PD1 antibody or isotype control (at 100µg/mouse) on days 6, 9, 12. Error bars represent SEM. *p<0.05, **p<0.01, ***p<0.001, ns, not significant, as determined by 2- way ANOVA (C) or log-rank test (D). VC A GzmB - PE H A - Single Cells - CD45 - CD3

- APC-CD8 FSC SSC PB FITC 후 Lymphocytes IFN - FSC-A FSC-A Aqua Live/Dead FITC-CD45 Alex APC-CD8

ATM KO GzmB - PE

H Single Cells - A - CD45 CD3 - - APC-CD8 FSC PB SSC 후 FITC Lymphocytes IFN - Aqua Live/Dead FITC-CD45 FSC-A FSC-A Alex APC-CD8

VC NK1.1 B - Single Cells PE H A - CD45 - - PB-CD3 FSC SSC FITC Lymphocytes CD4

FSC-A FSC-A Aqua Live/Dead - Alex PB-CD3

ATM KO NK1.1 - PE Single Cells H A - CD45 - - PB-CD3 FSC SSC FITC Lymphocytes CD4 FSC-A FSC-A Aqua Live/Dead - Alex

PB-CD3

Supplementary Figure 11. Additional data on lymphocyte infiltration into ATM deficient tumors. (A) Gating strategy and representative flow cytometry plots for the quantification of CD8+ T cells , GzmB+CD8+ and IFNγ+CD8+ T cells in vector control(VC) and ATM KO B16 tumors. (B) Gating strategy and representative flow cytometry graphs for the assessment of CD4+ T cells and NK cells in vector control(VC) and ATM KO B16 tumors. A B

Enrichment plot: GO_T_CELL_RECEPTOR_COMPLEX Enrichment plot: KEGG_T_CELL_RECEPTOR_SIGNALING_PATHWAY 0.7 0.45 0.6 0.40 0.5 0.35 0.30 0.4 0.25 0.3 NES = 2.0852757 0.20 0.2 0.15 NES = 1.7337495 0.1 p = 0 0.10 Enrichment score Enrichment score (ES) 0.0 0.05 p = 0.0017889087 Enrichment score Enrichment score (ES) 0.00

5.0 5.0 2.5 2.5 0.0 CD3D 0.0 CD8A -2.5 SKAP1 -2.5 PAK3 CD3E ITK 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 Ranked list metric(Signal2Notes)Rankedlist CD6 CD3D

CD3G metric(Signal2Notes)Rankedlist HRAS PTPN6 CD28 ZAP70 CD8A CARD11 PDCD1 CD247 LAT CD3E CD3G

Supplementary Figure 12. RNAseq profiling of ATM deficient tumors in vivo. Gene set enrichment analysis (GSEA) of T cell receptor complex (A) and T cell receptor signaling (B) gene expression in control and B16F10 ATMKO tumors. FDR calculated using GSEA. A B C *** *** *** *** 3000 3000 *** 3000 *** 500500 * * * * 3000 ** ** 400400 20002000 20002000 300300

200 10001000 200 10001000 tumor cells/mg #NK #CD4 cells/mg tumor cells/mg #CD4 #CD8 cells/mg tumor cells/mg #CD8 100 CD4 cells/mg tumor cells/mg CD4 NK cells/mg tumor cells/mg NK 100 CD8 cells/mg tumor cells/mg CD8

00 00 00 c c v O O O v O O O O c K K K K v O O O O DK D M DK DK D K K K M KO D g D T S K g S M DK D A B n AT A in T g ti TBK t A S n GA T S / /S A i c / /cG M /TBK M T M TM T A TM /cG ATM/ A A M T ATM/ A T A A ATM/St Supplementary Figure 13. Additional analysis of lymphocyte infiltration into ATM deficient tumors. Average numbers of tumor-infiltrating CD4+ T cells (A), CD8+ T cells (B), NK1.1+ NK cells (C) in tumors established from vector control (VC) , ATM KO B16F10, ATM/cGas DKO, ATM/TBK DKO, ATM/STING DKO B16F10 tumor cells inoculated in C57BL/6 mice. Flow cytometry analysis were done on day 13 post inoculation of 1x105 tumor cells. Error bars represent standard error of the mean (SEM). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant, as determined by 2-way ANOVA msk-overal

Mutation A 100 Mutation WT WT

50 Percent survival Percent Logrank P-Value: 0.3978 Overall Survival(%) 0 00 2020 4040 Number at risk Time Median 398 59 1 30.01 7176 1212 3 26.2 lung B Non-Small Cell Lung Cancer 100100 mt Mutation wt WT

5050 Survival(%) Percent survival Percent Logrank P-Value: 0.0931 00 00 2020 4040 6060 Monthsmonths Survival Number at risk Median 23 9 2 21 327 73 15 11

b-mt C 100100 Mutation-TMB/Low WT-TMB/Low b-wt

5050 Percent survival Percent p=0.0365 Overall Survival (%) Survival Overall 00 0 101020 30 40 50 MonthsMonths Survival Number at risk Median 11 8 5 3 1 NA 169 63 22 11 5 14

Supplementary Figure 14. Additional human clinical data regarding the influence of ATM mutations on tumor responses to ICB therapy. (A) Kaplan-Meier survival curve of patients from the whole MSK-IMPACT cohort with (red) or without (blue) ATM mutations who were not treated with ICB therapy. Data from the MSK-IMPACT clinical sequencing cohort (reference 43). (B) Kaplan-Meier survival data after immune checkpoint inhibitor therapy in non-small cell lung cancer patients with (red) or without (blue) ATM mutations. Data from the MSK-TMB cohort (reference 41). (C) Kaplan-Meier survival of curve of overall survival of patients with bladder cancer treated ICB therapy. Compared with Fig. 9D, only those patients with TMB<20 were considered to exclude the influence of MSI status. P values calculated by use of logrank test