Title Targeting p300 addiction in CBP-deficient cancers causes synthetic lethality by apoptotic cell death due to abrogation of MYC expression

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1 Supplementary Information Cancer Discovery Title Targeting p3 addiction in CBP-deficient cancers causes synthetic lethality by apoptotic cell death due to abrogation of MYC expression Authors Hideaki Ogiwara 1, Mariko Sasaki 1, Takafumi Mitachi 1, Takahiro Oike 1,2, Saito Higuchi 3, Yuichi Tominaga 3, and Takashi Kohno 1,* Author Affiliations 1 Division of Genome Biology, National Cancer Center Research Institute, Tokyo 14-45, Japan. 2 Department of Radiation Oncology, Gunma University Graduate School of Medicine, Gunma , Japan. 3 Oncology Research Laboratories, Daiichi-Sankyo Co., Ltd., Tokyo , Japan. Running Title Targeting p3 addiction in CBP-deficient cancers Keywords synthetic lethality; molecularly targeted therapy; epigenetics; paralog; p3; CBP; MYC 1

2 Contents: Supplementary Figure Legends Supplementary Fig. S1. Identification of the p3 gene as a synthetic lethal hit. Supplementary Fig. S2. Identification of MYC as a key factor in determining cancer cell survival under p3 depletion in CBP-KO cells or CBP depletion in p3-ko cells Supplementary Fig. S3. MYC transcriptional initiation is inactivated by suppression of histone H3K18/K27 acetylation upon p3 depletion in CBP-deficient cancer cells. Supplementary Fig. S4. p3 depletion is lethal in cancer cells harboring loss-offunction mutations in CBP. Supplementary Fig. S5. Response of CBP-deficient lung cancer cells to the p3 HAT inhibitor C646. Supplementary Fig. S6. Depletion or inhibition of p3 suppresses growth of hematopoietic cancers with loss-of-function mutation of CBP. Supplementary Fig. S7. Effect to BRD4 localization in the MYC locus in p3 depletion in CBP-deficient cancer cells. Supplementary Fig. S8. CBP/CREBBP and p3/ep3 aberrations and known druggable aberrations of FGFR1 or ERBB2 amplification in lung squamous cell carcinomas and stomach adenocarcinomas. Supplementary Tables Table S1. Raw data of survival fraction in sirna screen Table S2. The top13 statistically significant pathways obtained from WikiPathways analysis Table S3. Genes commonly involved in the 13 functional pathways Table S4. CBP and p3 mutations in a variety of cancers Table S5. Gene status of tumor cell lines Table S6. sirnas used in this study Table S7. Antibody for immunoblotting used in this study Table S8. TaqMan Gene Expression Assays used in this study Table S9. Primer used in this study 2

3 Supplementary Figure Legends Supplementary Fig. S1. Identification of the p3 gene as a synthetic lethal hit. (A B) p3 depletion in CBP-KO and CBP-deficient LK2 cells significantly decreased cell growth and survival., CBP-KO 2G2, and CBP-deficient LK2 cells were transfected with sirna for 48 h and then subjected to quantitative RT-PCR analysis (A), growth assay, or colony-formation assay (B). Expression levels of p3 and CBP were normalized against the level of GAPDH mrna in the same sample. The relative proliferation ratio or surviving fraction of sip3-treated cells at 7 days or 12 days after reseeding, respectively, was expressed as the percentage of cells transfected with targeting sirnas that proliferated or survived, relative to the corresponding percentage in cells transfected with non-targeting sirna. Data are expressed as means ± SD. (C, D) Depletion of p3 did not affect proliferation of non-cancerous cells. MRC5, HEK293T, and RPE-1 cells were transfected with sirna for 48 h and then assayed for immunoblot analysis (C) and cell proliferation (D). The fold change in proliferating ratio after reseeding is shown. Data are expressed as means ± SD. (E) Expression of CBP and p3 protein in, CBP-KO, and p3-ko cells. (F I) Cell growth inhibition by depletion of p3 or CBP. parental (CBP wt/p3 wt), CBP-KO (CBP-KO/p3 wt), or p3-ko (CBP wt/p3 KO) cells were transfected with sirna (, sip3 D1 or sicbp D2) for 48 h, and then subjected to quantitative RT-PCR analysis (F, H) or cell growth assay (G, I). The relative proliferating fraction of sirna-treated cells 7 days after reseeding is expressed as the fold change in the proliferation ratio after reseeding. Data are expressed as means ± SD. (J L) Cell growth following double depletion of p3 and CBP. (CBP wt/p3 wt) cells were transfected with sirna (, sip3 D1 and sicbp D2) for 48 h, and then subjected to quantitative RT-PCR analysis (J), cell growth assay (K), or colony-formation assay (L). The relative proliferating fraction of sirna-treated cells at 7 days after reseeding is expressed as the fold change in the proliferation ratio after reseeding. The relative surviving fraction of sirna-treated cells at 7 days or 12 days after reseeding is expressed as the percentage of cells transfected with targeting sirnas that survived, 3

4 relative to the corresponding percentage in cells transfected with non-targeting sirna. The numbers and sizes of colonies formed by cells subjected to CBP and/or p3 depletion are shown in the right panels. Data are expressed as means ± SD. (M, N) The proportion of G1-phase cells, but not apoptotic cells, increased upon depletion of CBP and p3 in cells. (CBP wt /p3 wt) cells were transfected with, or both sip3 D1 and sicbp D2, for 48 h. Two to eight days after reseeding, cell-cycle profiles and proportions of Annexin V positive apoptotic cells were assessed by flow cytometry. Data are expressed as means ± SD. Supplementary Fig. S2. Identification of MYC as a key factor in determining cancer cell survival under p3 depletion in CBP-KO cells or CBP depletion in p3-ko cells. (A) Genes downregulated by p3 depletion in CBP-KO cells or by CBP depletion in p3-ko cells. The top 1 ranking downregulated genes are shown for each condition. (B) Expression of genes involved in the cell cycle and apoptosis following p3 depletion. CBP wild-type (parental ) and CBP-KO ( 2G2) cells were subjected to quantitative RT-PCR 48 h after transfection with or sip3 (D1 and S1). The fold changes in gene expression relative to are shown. (C) Suppression of cell survival of CBP-KO cells by p3 depletion was rescued by exogenous expression of wild-type p3, but not HAT-defective p3. CBP-KO cells were transfected with or without a plasmid expressing p3 or HAT-defective p3 (HAT-) cdnas. Twenty-four hours later, cells were further transfected with sip3 (3 UTR: targeting the 3 UTR region of the p3 mrna) or. Forty-eight hours later (Day ), cells were seeded in 6-well plates. Cell survival was measured on day 12. Data are expressed as means ± SD. (D) Suppression of cell proliferation and survival of CBP-KO cells by p3 depletion was rescued by exogenous expression of MYC. CBP-KO cells with or without stable exogenous MYC cdna expression were transfected with or sip3 (D1 and S1). Forty-eight hours later (Day ), cells were seeded in 96- or 6-well plates. Cell viability was measured on day 5, and cell survival was measured on day 12. Immunoblot analyses of p3, MYC, and β-actin (loading control) at 48 h are shown. Data are expressed as means ± SD. 4

5 (E,F) Suppression of cell survival of HEK293T CBP-KO cells by p3 depletion was rescued by exogenous expression of MYC. HEK293T CBP-KO cells (as shown in Supplementary Fig. S2E) stably expressing or not expressing exogenous MYC cdna were transfected with or sip3 (D1). Forty-eight hours later (Day ), cells were seeded in 96-well plates. Cell viability was measured on day 5. Immunoblot analyses of p3, CBP, MYC and β- actin (loading control) at 48 h are shown. Data are expressed as means ± SD. (G) Transcriptional downregulation of several genes by p3 depletion in CBP-KO cells was rescued by exogenous expression of MYC. CBP-KO cells with or without stable exogenous MYC cdna expression were transfected with or sip3 (D1) and subjected to quantitative RT-PCR 48 h later. Fold changes in gene expression relative to are shown. (H) Suppression of proliferation of CBP-KO cells by p3 depletion was rescued by exogenous expression of MAX. CBP-KO cells were transfected with or without a plasmid expressing MAX cdnas and sirna (sip3 (D1) or ). Forty-eight hours later (Day ), cells were seeded in 96-well plates. Cell viability was measured on day 5. Immunoblot analyses of p3, MYC, MAX and β-actin (loading control). Data are expressed as means ± SD. Supplementary Fig. S3. MYC transcriptional initiation is inactivated by suppression of histone H3K18/K27 acetylation upon p3 depletion in CBP-deficient cancer cells. CBP and p3 proteins localized to the upstream region of the MYC locus. Quantitative genomic PCR was performed using input and ChIP DNAs. Relative ChIP enrichment values in the regions at the indicated distances from the transcription start site (TSS) in the MYC gene are expressed as percentages relative to the input DNA. Data are expressed as means ± SD. ChIP assays were performed on cells using control IgG or antibodies against CBP or p3. Supplementary Fig. S4. p3 depletion is lethal in cancer cells harboring loss-offunction mutations in CBP. (A, B) Synthetic-lethal effects assessed by cell-proliferation assay. Cells were transfected with sirna (, sip3 Dp) for 48 h, and then assayed for proliferation. The proliferating 5

6 fraction of sip3-treated cells after reseeding is expressed as the percentage of cells transfected with targeting sirnas that survived, relative to the corresponding percentage of cells transfected with non-targeting sirna (A). Data are expressed as means ± SD. Immunoblots showing p3 protein levels in cancer cells after sirnamediated knockdown (B). (C, D) Synthetic-lethal effects of sirnas against various targets of p3. H157 and SQ5 (CBP wild-type) cells and H173 and LK2 (CBP-deficient) cells were transfected with sirnas [ or sip3 (D1, S1, and D4)] for 48 h. After harvest, the cells were subjected to quantitative real-time PCR to detect p3 mrna. Expression was normalized against the level of GAPDH mrna (C). The surviving fraction of sip3-treated cells after reseeding is expressed as the percentage of cells transfected with targeting sirnas that survived, relative to the corresponding percentage of cells transfected with nontargeting sirna (D). Data are expressed as means ± SD. (E) Synthetic-lethal effects on cancer cells stably expressing p3-shrna. A549 and (CBP wild-type) cells and LK2 and H52 (CBP-deficient) cells were infected with shrna (shp3 or shluc)-expressing lentiviral vectors and selected for 2 weeks in medium containing puromycin. The cells were then harvested and subjected to immunoblot analysis and colony-formation assays. The percentage of shp3-expressing cells surviving 12 days after reseeding, relative to the corresponding percentage of shluc cells, is shown. Immunoblots were probed with antibodies against p3 and α-tubulin (loading control). Data are expressed as means ± SD. (F) Increased expression of apoptotic markers following depletion of p3 in CBPdeficient cancer cell lines. H157 (CBP wild-type) cells and LK2 (CBP-deficient) cells were transfected with sirna (, sip3 Dp) for 48 h and then subjected to immunoblot analysis. Data are expressed as means ± SD. Immunoblot analysis was performed using antibodies against p3, cleaved PARP (a marker of apoptosis), p21 (a marker of senescence), the degraded form of LC3B (a marker of autophagy; arrowhead), and β- actin (loading control). (G) Suppression of survival of LK2 CBP-deficient cancer cells by p3 depletion was rescued by exogenous expression of MYC. LK2 cells were transfected with or without a plasmid expressing MYC cdna and sirna ( or sip3 (D1)). Forty-eight hours later 6

7 (day ), cells were seeded in 96-well plates. Cell viability was measured on day 5. Immunoblot analyses of p3, MYC, and β-actin (loading control) are shown. Data are expressed as means ± SD. (H K) Induction of apoptosis by shrna-mediated p3 depletion in CBP-deficient lung cancer cells. -shnt, -shp3, LK2-shNT, and LK2-shp3 cells were cultured in the presence or absence of doxycycline (Dox) for 12 h before analysis. (H) Immunoblot analysis of p3 and β-actin expression and cell proliferation, as determined by ATP-based cell viability assay. (I) Surviving fraction of Dox-treated or - untreated cells is expressed as the percentage of shrna-expressing cells that survived, relative to the corresponding percentage of cells not expressing shrna (number of colonies formed by Dox-treated cells/number of colonies formed by Dox-untreated cells). (J) Induction of apoptosis by depletion of p3. Cells were harvested and subjected to flow cytometry to determine the cell-cycle profile and proportion of Annexin V positive apoptotic cells. Data are expressed as means ± SD. (L) In vivo growth of tumor cells expressing non-targeting shrna immediately after their injection. -shnt cells and LK2-shNT cells were implanted subcutaneously into BALB/c-nu/nu mice. The mice were then randomly divided into two groups: one group was fed a diet containing Dox (Dox+), and the other was fed a control diet (Dox-). Tumor volumes in each group are shown. (M) In vivo growth of tumor cells expressing non-targeting shrna after engraftment. LK2-shNT cells were implanted subcutaneously into BALB/c-nu/nu mice. When the tumors reached more than 2 mm 3, mice were randomly divided into two groups: one group was fed a diet containing doxycycline (Dox) (Dox+), and the other was fed a control diet (Dox-). Changes in tumor volume in both groups are shown. Data are expressed as means ± SE. (N) Suppression of orthotopic tumor growth in vivo by p3 depletion in CBP-deficient LK2 cells. LK2-shp3 lung cancer cells following Dox and mock treatment were implanted into the thoracic cavity of four BALB/c-nu/nu mice, respectively. The former four mice were fed a diet containing Dox (Dox+), and the latter four mice were fed a control diet (Dox-). Twenty-four days after implantation, mice were sacrificed. The four mice in both groups developed orthotopic xenografts (two mice with intrathoracic 7

8 xenografts and two mice with extrathoracic xenografts in both groups). Left; A marked deference in tumor volumes was observed between the two groups. Data are expressed as means ± SE. Right; Pictures of intrathoracic xenografts in mice fed a control diet (Dox-) or a diet containing Dox (Dox+). Supplementary Fig. S5. Response of CBP-deficient lung cancer cells to the p3 HAT inhibitor C646. (A) Survival after treatment with C646 in CBP-deficient or CBP wild-type cancer cells. Cells were subjected to a colony-formation assay in the presence or absence of C646. Data are expressed as means ± SD. (B) Induction of apoptotic cells by C646 treatment. CBP wild-type A549 and cells and CBP-deficient H52 and H173 cells were treated with C646 [ µm (C646-) or 15 μm (C646+)] for 48 h. The proportions of Annexin V positive apoptotic cells were determined by flow cytometry. (C) Expression of MYC in cells treated with C646. CBP wild-type, H661, and A549 cells were subjected to quantitative RT-PCR analysis 48 h after treatment with the indicated concentrations of C646. The fold change in expression of MYC is expressed relative to the level in non-treated cells. (D) MYC protein expression in cells treated with C646. CBP-deficient H52 and H173 cell lines were treated with C646 [ µm (C646-) or 15 μm (C646+)] for 48 h, harvested, and subjected to immunoblot analysis with antibodies against MYC, acetylated histone H3 K18 (H3K18ac), K27 (H3K27ac), and β-actin (loading control). (E) Suppression of transcriptional initiation of MYC by impairment of histone acetylation upon C646 treatment in CBP-deficient H173 cells. The transcription start site (TSS) and promoter region (Pro) are indicated. Acetylation of Histone H3K27 and localization of RNA polymerase II (RNAPII) at the MYC locus 48 h after treatment with or without 15 μm C646 are shown. (F) Body weights of mice bearing LK2 xenografts and treated or not treated with C646. (G) Human small-cell lung cancer (SCLC) cell lines used to examine sensitivity to C646. Data regarding missense and deleterious (Del) mutations were obtained from the 8

9 cbioportal and COSMIC databases. Functional impact values (neutral, low, medium, and high) of missense mutations were obtained from the MutationAssessor database. (H) Cell viability of CBP-deficient or CBP wild-type SCLC cells after treatment with C646. Cells were subjected to cell-proliferation assay in the presence or absence of C646 for 96 h. Data are expressed as means ± SD. (I) Survival CBP-deficient or CBP wild-type lung cancer cells after treatment with L2, SGC-CBP3, or I-CBP112. Cells were subjected to a colony-formation assay in the presence or absence of drug treatment. Data are expressed as means ± SD. Supplementary Fig. S6. Depletion or inhibition of p3 suppresses growth of hematopoietic cancers with loss-of-function mutation of CBP. (A) Data from cancers with predicted high-impact (loss-of-functional) mutations, obtained from the International Cancer Genome Consortium (ICGC) ( (B) Cell viability of CBP-deficient or CBP wild-type hematopoietic cancer cells after treatment with C646. Cells were subjected to cell-proliferation assay in the presence or absence of C646 for 96 h. Data are expressed as means ± SD. (C) Cell viability of CBP-deficient or CBP wild-type hematopoietic cancer cells after treatment with SGC-CBP3 or I-CBP112. Cells were subjected to cell-proliferation assay in the presence or absence of SGC-CBP3 or I-CBP112 for 72 h. Data are expressed as means ± SD. (D) Induction of apoptotic cells by C646 treatment. CBP wild-type U2932 and RC-K8 cells and CBP-deficient WSU-NHL, VAL, and SU-DHL-5 cells were treated with or without 1 μm C646 for 48 h, and then assayed by flow cytometry to determine the proportion of Annexin V positive apoptotic cells. (E) p3 depletion is lethal in CBP-deficient hematopoietic cancer cells. Before analysis, Jurkat-shNT and Jurkat-shp3 cells were cultured in the presence or absence of doxycycline (Dox). Cell proliferation was assessed in a cell-counting assay. Data are expressed as means ± SD. (F) Tumor growth in vivo after injection of tumor cells treated with non-targeting shrna into tumor-engrafted mice. Jurkat-shNT cells were implanted subcutaneously into BALB/c-nu/nu mice. When the tumors reached more than 2 mm 3, the mice were 9

10 randomly divided into two groups and fed a diet containing doxycycline (Dox) (Dox+) or a control diet (Dox-). Changes in tumor volume in both groups are shown. Data are expressed as means ± SD. Supplementary Fig. S7. Effect of p3 depletion on BRD4 localization at the MYC locus in CBP-deficient cancer cells. ChIP assay was performed on CBP-deficient LK2 cells to examine the localization of BRD4 at the MYC locus after transfection with or sip3 (Dp). Quantitative genomic PCR was performed using input and ChIP DNAs. Relative ChIP enrichment values in the regions at the indicated distances from the transcription start site (TSS) in the MYC gene are expressed as percentages relative to the input DNA. Data are expressed as means ± SD. ChIP assays were performed using control IgG or an antibody against BRD4. Supplementary Fig. S8. CBP/CREBBP and p3/ep3 aberrations and known druggable aberrations of FGFR1 or ERBB2 amplification in lung squamous cell carcinomas and stomach adenocarcinomas. Data from 178 cases of squamous cell lung carcinoma and 287 cases of stomach adenocarcinomas are shown (Supplementary Table S4; Cancer Genome Atlas [TCGA] data from Red: high-impact mutation; blue: other missense mutation; orange: gene amplification; gray: no alteration. 1

11 A C Relative mrna Expression p3 β Actin KD Efficiency sitarget sitarget 2G2 (KO) sip3 sitarget LK2 (mut) MRC5 HEK293T RPE1 sip3 sip3 HDAC8 DOT1L SIRT4 SMARCA1 KDM4B EP3 D Relative Proliferation B Proliferation (%) MRC5 Supplementary Fig. S sihdac (Day) Cell Growth sidot1l sisirt4 sismarca1 RPE-1 sikdm4b siep (Day) Survival (%) HEK293T Colony Formation sihdac (Day) sidot1l sip3 sisirt4 E sismarca1 sikdm4b siep3 CBP p3 CBP p3 β-actin (wt) 2G2 (KO) LK2 (mut) wt KO wt wt wt KO Survival (%) Colony Formation sip3+sicbp F H J Relative Expression Relative Expression CBP p3 p3 mrna 1.2 CBP mrna p3 wt KO CBP wt wt sip3 sicbp G I K Relative Proliferation Relative Proliferation Cell Growth (Day) Cell Growth (Day) L M N CBPwt p3wt Relative Expression sip3 +sicbp wt KO wt wt CBP mrna p3 mrna Cell Cycle (%) Day CBPwt p3wt sip3 +sicbp Relative Proliferation Cell Cycle Profile Cell Growth (Day) G1 S G2/M subg1 CBPwt CBPwt sip3 CBPKO CBPKO sip3 p3wt p3wt sicbp p3ko p3ko sicbp CBPwt p3wt sip3+sicbp AnnexinV+ cells (%) Apoptosis CBPwt p3wt sip3 +sicbp (Day)

12 A CBPKO: Down-regulated genes by p3 knockdown Supplementary Fig. S2 Top Ranking CDC45L CDC45L CCNA2 CCNA2 E2F7 CCND1 RFC3 CCND1 PMAIP1 RAD51 PLAU TYMS FANCD2 2 MCM1 E2F2 CDC45 CDC45 MCM4 E2F1 DSCC1 eif-4ebp BIRC5 BLM WNT16 DNMT3L BRCA1 3 CDT1 ORC1L E2F2 E2F2 UBE2C H2AFX CHTF18 EGF BIRC3 CCND1 FOSL1 MTHFD1L H2AX 4 RFC3 MCM2 MCM1 CDT1 KIF15 CDC25A2 UBE2C NRG2 TRAF1 E2F1 CCND1 DHFR/MTHFD1L CDK2 5 ORC1L MCM7 EP3 RFC3 Histone H4 CDK6 RFC4 NRG1 TNFRSF21 MYC WNT1A DNMT1 Cyclin B 6 MCM2 MCM5 PMYT1 ORC1 MYBL2 c-myc FANCI Myc/MYC HELLS BRCA1 LDLR MTHFD2 MDC1 7 MCM7 CCND1 MD2L1 MCM7 TPX2 RAD52 UBE2T NRG3 CASP1 PLK1 c-myc/myc PSAT1 MRE11 8 MCM5 MCM4 ORC1 TYMS TOP2A CREB1 TOP2A PI3K MYC MPIP1 WNT1B SHMT2 Chk2 9 MCM4 E2F1 MCM2 CCND1 ECT2 TP53 MYC BTC TRAF2 CDC25A WNT5B CBS Chk1 1 PCNA PCNA MCM7 MCM4 AURKA ATM BRIX1 PDK1 IGF2 CDK2 RAC3 MTHFD1 GADD45A B p3ko: Down-regulated genes by CBP knockdown Top Ranking MCM1 CDC45L CCNA2 CCNA2 UBE2C E2F1 UBE2C CCND1 BIRC5 PLK1 PLAU TYMS Cyclin B 2 CDC45L E2F2 MCM1 TYMS TOP2A CDC25A2 TOP2A NRG2 PMAIP1 BLM FOSL1 DHFR/MTHFD1L FANCD2 3 CDT1 MCM6 CDC45 CDC45 E2F7 CCND1 UBE2T Myc/MYC HELLS BRCA1 CCND1 DNMT1 BRCA1 4 MCM6 PRIM1 CDC2 TOP2A CENPF H2AFX FANCI eif-4ebp TNFRSF25 RAD51 c-myc/myc MTHFD1L H2AX 5 PRIM1 CDK1 E2F2 E2F2 KIF15 CDK6 CHTF18 NRG1 MYC E2F1 RAC3 DNMT3L CDK2 6 ORC1L ORC1L PLK1 CDT1 ECT2 c-myc DSCC1 EGF TNFRSF21 TAK1L MAPK9 GCLM MDC1 7 MCM7 MCM7 MCM6 HMGB2 MYBL2 TP53 RFC4 HBEGF BCL2 BCL2 BBC3 DNMT3B (MDM4) 8 MCM2 CCNB1 MD2L1 MCM6 TPX2 RAD52 RFC3 ERBB1 CASP2 MPIP1 NFKB2 PSAT1 Chk1 9 PCNA MCM2 CDK1 RRM2 Histone H4 ATM LMNB2 p53 FAS CDC25A WNT1A MTHFD2L GADD45A 1 MCM5 PCNA ORC1 PRIM1 AURKA CCND3 MYC EREG CASP1 CCND1 CDKN2A MAT2A SMC1A Relative Expression CBP mrna mrna Expression wt KO wt KO wt KO wt KO wt KO wt KO wt KO wt KO wt KO wt KO wt KO wt KO wt KO wt KO wt KO wt KO EP3 MYC CCND1 CDK4 CDK6 CCNA2 CCNE1 CCNE2 CDKN1A BIRC5 XIAP BCLxl BCL2 MCL1 BIM BAX sip3 D1 sip3 S1 C Survival (%) E G Colony Formation HEK293T CBP status wt KO CBP β actin Relative Expression EP3 mrna vec MYC F p3 MYC β actin sip3_3'u sip3 D HEK293T CBPKO vec MYC MYC mrna vec MYC sip3 MYC p3 MYC β-actin Proliferation (%) CBP KO sip3 D1 S1 CCND1 mrna vec MYC Cell Growth vec 2 1 MYC CDC45 mrna vec MYC Proliferation (%) sip E2F2 mrna vec MYC Cell Growth vec H p3 MYC MAX β actin MYC sip3 D1 sip3 S1 CBPKO vec MAX sip3 E2F1 mrna vec MYC sip Survival (%) Proliferation (%) vec Colony Formation vec MYC MAX mrna MYC Cell Growth vec MAX CBPKO sip3 sip3 D1 sip3 S1 sip3

13 Supplementary Fig. S3 CBP ChIP p3 ChIP Input (%) Distance from TSS of MYC gene (kb) H157 IgG H157 CBP Input (%) Distance from TSS of MYC gene (kb) H157 IgG H157 p3 Input (%) CBP ChIP Distance from TSS of MYC gene (kb) H52 IgG H52 CBP Input (%) p3 ChIP Distance from TSS of MYC gene (kb) H52 IgG H52 p3

14 EP3 mrna sip3 D1 sip3 S1 sip3 D4 CBP wt A549 shluc shp3 F LK2 H52 A549 LK2 H52 LK2 Cell Proliferation LK2 4 2 sip3 vec MYC p3 clevparp p21 LC3B CBP mut sip3 β actin G p3 β actin LK2 p3 p3 MYC β actin β actin LK2 CBPmut shnt shp3 J L 4 5 Day shnt DoxDox Day 6 8 Survival (%) CBPwt LK2 CBPmut 6 shnt Dox shnt Dox + shp3 Dox shp3 Dox Colony Formation K LK2 CBP wt Apoptosis shnt DoxshNT Dox+ shp3 Doxshp3 Dox+ LK2 CBP mut CBP wt CBP mut Day LK2 shnt 15 DoxDox Day M Tumor volume (mm3) 8 Tumor volume (mm3) Relative Proliferation I Relative Proliferation Dox p3 β actin AnnexinV+ (%) CBPwt shnt shp3 LK2 shnt DoxDox Day N Tumor Volume (mm3) H sip3 sip3 TE1 sip3 H173 sip3 sip3 sip3 sip3 sip3 sip3 sip3 LK2 TE8 MYC sip3 vec CBPmut H29 H148 H287 KM12C H52 sip3 H322 sip3 LoVo Tumor volume (mm3) CBP mut H157 HCT116 sip3 HT29 sip3 sip3 SW48 SW62 CBP wt p3 αtubulin CBP wt sip3 sip3 SQ5 sip3 H157 sip3 H661 sip3 CBP mut Colony Formation sip3 sip3 D1 sip3 S1 sip3 D4 sip3 Survival (%) Colony Formation E CBPwt A sip H157 LK2 H Survival (%) 4 Proliferation (%) B 2 sh Lu sh c p3 sh Lu sh c p3 sh Lu sh c p3 sh Lu sh c p3 Cell Lines A549 H661 H157 SQ5 CBP proficient SW48 SW62 HT29 HCT116 LoVo H322 H29 CBP Low/Medium H148 impact mutation H287 KM12C H52 TE8 CBP Deleterious or High impact LK2 mutation H173 TE1 D H157 LK2 H173 Proliferation (%) Relative mrna Expression A Supplementary Fig. S4 C Orthotopic Xenograft DoxDox+ 5 LK2 shp3 LK2 shp3 Intrathoracic Xenograft Dox+ Dox-

15 Supplementary Fig. S5 A Survival (%) C646 sensitivity C646 (μm) H661 A549 H157 H322 H52 LK2 H173 B AnnexinV+ cells (%) C646- C646+ A549 H52 H173 CBPwt Apoptosis CBPmut C Relative Expression MYC mrna H661 A549 CBP wt μm 15 μm 2 μm 3 μm D G I C646 Myc β actin H3K18ac H3K27ac Survival (%) H52 CBP proficient H173 CBP Low impact mutation CBP Deleterious mutation E Cell Lines L2 Sensitivity.5 1 L2 (μm) Input (%) H3K27ac pro TSS Distance from TSS in MYC gene (kb) CBP Status Mutation H157 A549 H661 H52 H173 LK2 C646- C646+ Functional Impact or Deletion Survival (%) Input (%) RNAPII pro TSS Distance from TSS in MYC gene (kb) p3 Status Mutation SGC-CBP3 Sensitivity SGC-CBP3 (μm) C646- C646+ Functional Impact or Deletion H526 wt - wt - HCC33 A1824T Low wt - H2122 N83T Low wt - H29 HD Del wt - H157 A549 H661 H52 H173 LK2 F Body wight (g) H Proliferation (%) Survival (%) Mouse with LK2 Xenograft Day C646 Sensitivity Mock C C646 (μm) I-CBP112 Sensitivity I-CBP112 (μm) H526 HCC33 H2122 H29 H157 A549 H661 H52 H173 LK2

16 A AnnexinV+ cells (%) C646- C646+ U2932 RCK8 CBPwt Apoptosis WSU NHL VAL CBPmut SU DHL5 Cell Number (x 1 5 cells/ml) Supplementary Fig. S6 Cancer with High Impact mutation of CBP Site Tumour Type Tumour Subtype Percentage of donors with the CREBBP mutated gene Blood Malignant Lymphoma Germinal center B-cell derived lymphomas 18.2% Head and neck Thyroid cancer Papillary thyroid carcinoma 13.3% Bladder Bladder cancer Urothelial carcinoma 11.7% Stomach Gastric cancer Adenocarcinoma 1.% Uterus Endometrial cancer Uterine corpus endometrial carcinoma 8.9% Lung Lung cancer Squamous cell carcinoma 7.9% Skin Skin cancer Cutaneous melanoma 6.3% Cervix Cervical cancer Cervical squamous cell carcinoma 6.2% Esophagus Esophageal cancer Squamous carcinoma 5.7% Colorectal Rectal cancer Adenocarcinoma 5.% Colorectal Colon cancer Adenocarcinoma 4.6% B D Proliferation (%) C646 sensitivity C646 (μm) U2932 Loucy RC-K8 RL Farage WSU-NHL VAL Jurkat SUDHL5 SUDHL6 E C Proliferation (%) SGC-CBP3 Sensitivity SGC-CBP3 (μm) Cell Proliferation (Day) Proliferation (%) shnt DoxshNT Dox+ shp3 Doxshp3 Dox+ F I-CBP112 Sensitivity I-CBP112 (μm) Tumor volume (mm 3 ) Jurkat shnt Day U2932 RCK8 RL Jurkat SUDHL5 SUDHL6 Dox- Dox+

17 Supplementary Fig. S7 Input (%) BRD4 ChIP sip Distance from TSS of MYC gene (kb)

18 Supplementary Fig. S8 Lung Squamous Cell Carcinoma (TCGA, Nature 212) 178 samples CBP p3 FGFR1 ERBB2 Stomach Adenocarcinoma (TCGA, Nature 214) 287samples CBP p3 ERBB2 High impact mutation low impact mutation Amplification No alteration

19 Table S1. Raw data of survival fraction in sirna screening MRC5 Survival (%) STD CHMP4B ARID1B HDAC SMARCB SMARCD CHD KDM4A YY MLL SMYD SRCAP SMYD MLL HDAC ARID1A SIRT SMYD INO CHD HLTF SMARCC RAD54L BRD JMJD CHD UTX BRD SETD HELLS PARP HDAC EP

20 KAT2A CHD UCHL BANF KDM4D SIRT SETD KDM6B MLL TRRAP CHMP NSD HDAC SMARCD RUVBL RUVBL KAT6B KDM3B SIRT LOC MYST KDM5B HDAC BRD CHRAC KDM2B ACTL6A ERCC JMJD1A HDAC SMARCE SMARCAD SIRT HDAC SETD1B

21 HMG2B SMARCA BRD BPTF BRD ARID CHD CLOCK MYST HDAC SMARCC POLE SETD1A TFPT JARID1C SMARCAL RBBP CHD BRD JMJD2C BATF SUV42H MYST AOF SETDB CHMP4C SUV39H KAT NCOA PBRM SUV39H CHD SNF HDAC SETDB

22 EP JARID1D CHD BAZ1A ASH1L DPF KDM5A KAT2B NCOA KDM1B ACTL6B ELP CSRP2BP SIRT CDK SMARCA SHPRH EHMT SMARCA BRD SIRT SUV42H SMARCD HDAC CREBBP CHD SMARCA HAT CDK CDK MLL KDM4B SIRT HDAC NCOA

23 DOT1L HFL1 Survival (%) STD CHMP4B ARID1B CHD HDAC HELLS INO ARID1A SMARCB MLL RAD54L CHMP HLTF KDM4A BRD CHD SMARCD BRD SMARCC MLL SMYD SMYD SIRT KAT6B SRCAP SMYD YY MLL SMARCC SETD UTX

24 HMG2B PARP HDAC RUVBL KDM5B LOC EP CHD KDM2B NSD CHD RUVBL CLOCK UCHL KAT2A SIRT SMARCD SUV39H ACTL6A SIRT KDM6B SIRT KDM4D BANF HDAC HDAC JMJD RBBP DPF CHD SETD1A SMARCE HDAC BRD BRD

25 BRD KDM5A SETD KAT HDAC ERCC TRRAP MYST CHRAC SMARCAL SMARCAD CHD BATF SMARCA CHMP4C JMJD1A SUV39H JMJD2C CDK HDAC ARID HDAC BPTF CSRP2BP SUV42H MYST SETDB KDM3B SETD1B POLE ELP SMARCA MYST SMARCA JARID1C

26 KAT2B ASH1L EHMT CHD CREBBP HDAC NCOA HDAC SNF TFPT SETDB SUV42H BRD SHPRH AOF KDM1B ACTL6B CDK NCOA BAZ1A PBRM EP CHD JARID1D SMARCA BRD HAT SIRT CHD SMARCD SIRT CDK SIRT KDM4B DOT1L

27 NCOA HDAC MLL A549 Survival (%) STD RUVBL YY SRCAP CHD RUVBL CHD CHD SMARCC HLTF SMARCD RAD54L EP ARID1B BRD ARID SETD RBBP INO CHMP POLE BANF ERCC HELLS TFPT HMG2B HDAC CHMP4B ARID1A

28 HDAC LOC ACTL6B SETD1B HDAC SUV39H CHD BRD KAT6B SMYD HDAC TRRAP SMARCD SUV39H CHRAC SMARCB BRD UTX UCHL BATF KDM6B ACTL6A MLL SMYD MLL PARP SNF BRD JARID1C MLL KDM4A CSRP2BP HDAC CHD CHD

29 DOT1L BRD KDM2B SIRT SIRT SIRT MLL NCOA DPF SMARCE KDM5A KDM3B KAT2A CHD BRD SMARCD SMARCAD CHD PBRM HDAC BPTF SETD1A CDK AOF SMARCC SMARCAL SMARCA SMARCA SMYD ELP KDM5B CLOCK HDAC BAZ1A CDK

30 HDAC SETDB MYST SUV42H JMJD1A SMARCA SUV42H KDM4D BRD HDAC CHMP4C HAT KDM1B CHD SIRT SHPRH JARID1D CREBBP NSD ASH1L KAT NCOA SMARCA HDAC JMJD2C EHMT JMJD SETDB MYST KAT2B SIRT HDAC KDM4B NCOA CDK

31 SIRT SIRT MYST SETD EP LK2 Survival (%) STD MLL INO KDM4A SMARCB CHD HDAC CHD HDAC HDAC ARID1B RUVBL SUV39H SMYD MLL HELLS HDAC BRD SETD ERCC SMARCA SUV39H HDAC TFPT SIRT KDM5B MYST

32 SMARCAD RUVBL SRCAP SMARCC RAD54L KDM4B HDAC MLL BPTF UTX AOF JARID1C HLTF SMARCAL EP KAT6B CHMP4B SMARCE CHD HDAC SIRT HDAC SETD1A KDM5A YY CHD ARID1A SMARCD SETD1B BRD SMYD CSRP2BP HDAC SMARCD CHMP

33 CHD SMARCA POLE HMG2B BRD RBBP DOT1L SIRT BRD SIRT CHMP4C SMYD CREBBP UCHL CHD KDM2B ACTL6A KAT ELP SMARCC CHD CLOCK SHPRH EHMT SMARCA EP SIRT ASH1L KDM4D BRD LOC SUV42H SIRT HDAC CDK

34 NCOA CDK MYST KAT2B SETDB MLL KDM1B KDM3B KAT2A NSD MYST KDM6B CDK CHRAC ACTL6B JMJD1A JARID1D NCOA SUV42H BATF SMARCA BANF ARID SETD SIRT TRRAP SMARCD DPF JMJD JMJD2C HAT CHD SETDB NCOA SNF

35 PARP BAZ1A BRD PBRM HDAC CHD BRD

36 Table S2. The top 13 statistically significant pathways obtained from WikiPathways analysis Rank Pathway p-value 1 Hs_DNA_Replication_WP466_ E-26 2 Hs_G1_to_S_cell_cycle_control_WP45_ E-11 3 Hs_Cell_Cycle_WP179_ E-1 4 Hs_RB_in_Cancer_WP2446_ E-1 5 Hs_Gastric_cancer_network_1_WP2361_ E-1 6 Hs_miRNAs_involved_in_DNA_damage_response_WP1545_ E-6 7 Hs_Gastric_cancer_network_2_WP2363_ E-5 8 Hs_ErbB_Signaling_Pathway_WP673_ E-5 9 Hs_Apoptosis_WP254_ Hs_Integrated_Breast_Cancer_Pathway_WP1984_ Hs_DNA_Damage_Response_(only_ATM_dependent)_WP71_ Hs_Trans-sulfuration_and_one_carbon_metabolism_WP2525_ Hs_ATM_Signaling_Pathway_WP2516_ Functional characterization of 1,936 genes differentially expressed in both p3-depleted CBP-KO cells or CBP-depleted p3-ko cells by pathway analysis. The top 13 statistically significant pathways obtained from WikiPathways analysis (p <.1; Fig. 2A) are shown. Table S3. Genes commonly involved in the 13 functional pathways Fold change relative to sample Overlapping Pathways Gene Name [CBPKO sip3] [p3ko sicbp] [wt sip3] [wt sicbp] 5 MYC CCND CDC E2F E2F CDT UBE2C MCM MCM MCM BRCA CCNA ORC1L TOP2A TYMS Genes commonly involved in the 13 functional pathways. Genes overlapping in the functional pathways are also shown, and their -fold change in expression level upon p3 or CBP depletion relative to, are shown as log2-transformed values.

37 Table S4. CBP and p3 mutations in a variety of cancers No. Study* No. of cases examined for mutation Percentage of cancers with CBP mutaions CBP mut/ p3 wt No. of cases CBP mut/ p3 mut CBP wt/ p3 mut CBP wt/ p3 wt 1 Bladder Urothelial Carcinoma (BGI, Nature Genetics 213) Small Cell Lung Cancer (CLCGP, Nature Genetics 212) Colorectal Adenocarcinoma (Genentech, Nature 212) Bladder Urothelial Carcinoma (TCGA, Nature 214) Uterine Corpus Endometrioid Carcinoma (TCGA, Nature 213) Colorectal Adenocarcinoma (TCGA, Nature 212) Lung Squamous Cell Carcinoma (TCGA, Nature 212) Lung Adenocarcinoma (Broad, Cell 212) Head and Neck Squamous Cell Carcinoma (Broad, Science 211) Adenoid Cystic Carcinoma (MSKCC, Nature Genetics 213) Head and Neck Squamous Cell Carcinoma (TCGA, in revision) Skin Cutaneous Melanoma (Broad, Cell 212) Small Cell Lung Cancer (Johns Hopkins, Nature Genetics 212) Lung Adenocarcinoma (TCGA, Nature, in press) Liver Hepatocellular Carcinoma (AMC, Hepatology in press) Skin Cutaneous Melanoma (Yale, Nature Genetics 212) Glioblastoma (TCGA, Cell 213) Medulloblastoma (PCGP, Nature 212) Ovarian Serous Cystadenocarcinoma (TCGA, Nature 211) Esophageal Adenocarcinoma (Broad, Nature Genetics 213) Pancreatic Adenocarcinoma (ICGC, Nature 212) Prostate Adenocarcinoma (Broad_Cornell, Cell 213) Breast Invasive Carcinoma (British Columbia, Nature 212) Medulloblastoma (Broad, Nature 212) Kidney Renal Clear Cell Carcinoma (BGI, Nature Genetics 212) Breast Invasive Carcinoma (Broad, Nature 212) Medulloblastoma (ICGC, Nature 212) Kidney Renal Clear Cell Carcinoma (TCGA, Nature 213) Multiple Myeloma (Broad, Cancer Cell 214) Breast Invasive Carcinoma (TCGA, Nature 212) Total** * Studies published or in press, in which CBP muations were detected at least in a case, are picked up. ** P-values for mutually exclusiveness between CBP and p3 mutations assessed by Fisher's exact test is 1x 1-7. Total Table S5. Gene status of tumor cell lines Gene Status Tissue Cell Lines CBP p3 SMARCA4 SMARCA2 ARID1A ARID1B SMARCB1 PBRM1 ARID2 EZH2 SETD2 SETBP1 KAT6B Lung HFL1 wt wt wt ND wt ND ND wt wt wt wt wt ND Lung MRC5 wt wt wt ND wt ND ND wt wt wt wt wt ND Lung A549 wt wt Q729fs wt wt wt wt wt wt wt wt wt wt Lung wt wt D578/K586del fs wt wt wt wt wt wt wt wt ND wt Lung H661 wt wt L1161fs wt wt wt wt ND ND wt ND ND wt Lung H157 wt wt T58fs P57fs wt wt wt wt wt wt ND K197I ND ND Lung SQ5 wt wt wt wt wt wt wt ND ND ND ND ND ND Colon SW48 wt wt wt ND wt wt wt ND ND wt ND ND E114dup Colon SW62 wt P144L wt ND wt wt wt ND ND wt ND ND E114dup Colon HT-29 wt M147fs wt ND wt wt wt ND ND wt ND ND wt Colon HCT116 wt K1648fs L1163P wt wt wt wt wt wt wt G249R ND T1525I Colon LoVo T573 wt wt ND F2141Sfs wt wt ND ND wt ND ND wt Lung H322 S893L wt ND ND ND ND ND ND ND ND ND ND ND Lung H29 H67R wt wt ND wt wt wt ND ND wt ND ND E1151* Lung H148 S168del wt wt ND wt wt wt ND ND wt ND ND wt Lung H287 I1846M D1485splice wt ND wt wt wt ND ND wt ND ND wt Colon KM12C R1664C P1452L/ Q761R P197S wt wt wt wt wt wt wt wt ND N83Tfs*5 Lung H52 R1446C wt wt wt wt wt wt ND ND wt wt ND wt Esophagus TE8 Q1765fs wt wt ND wt wt wt ND ND wt ND ND wt Lung LK2 HD ex3 wt wt wt wt wt wt wt wt wt wt wt wt Lung H173 W1472C/N2175S A1437V D668/N758del fs wt wt wt wt ND ND wt wt ND wt Esophagus TE1 Y1539C W159C wt ND wt wt wt ND ND wt ND ND wt Cell lines used in screening assay CBP wild type CBP Low impact mutation CBP Deleterious or High impact mutation

38 Table S6. sirna used in this study Target gene sirna name Catalog number Maker Non-targeting D Dharmacon p3 sip3 Dp L Dharmacon p3 sip3 D1 J Dharmacon p3 sip3 D2 J Dharmacon p3 sip3 D3 J Dharmacon p3 sip3 D4 J Dharmacon p3 sip3 U1 DHAPB-264 Dharmacon p3 sip3 U2 DHAPB-455 Dharmacon p3 sip3 S1 Hs_EP3_2818 Sigma CBP sicbp Dp L Dharmacon CBP sicbp D2 J Dharmacon CBP sicbp S1 Hs_CREBBP_39 Sigma Table S7. Antibody for immunoblotting used in this study Target Host Cat# Maker MW Dilution CBP Rabbit sc-369x Santacruz 3 1 CBP Rabbit 7389 CST 3 1 p3 Mouse sc-48343x Santacruz 3 1 p3 Rabbit A3-358 Bethyl 3 5 c-myc Rabbit 565 CST 55 1 H3K18ac Rabbit Millipore 17 3 H3K27ac Rabbit 8173 CST 17 3 βactin Rabbit 497 CST 45 1 cleaved PARP Rabbit 5625 CST 89 1 p21 Rabbit 2947 CST 21 1 LC3B Rabbit 3868 CST 14,16 1 CCND1 Mouse 2926 CST 36 1 CDC45 Rabbit GTX11586 GeneTex 66 1 MAX Rabbit A32-866A Bethyl 18 2 Table S8. TaqMan Gene Expression Assays used in this study Target Cat# p3 Hs914223_m1 CBP Hs231733_m1 MYC Hs15348_m1 CCND1 Hs765553_m1 BCL2 Hs6823_m1 CDK6 Hs126371_m1 CDK4 Hs _g1 MCL1 Hs _g1 BCLXL Hs236329_m1 CCNE1 Hs126536_m1 CCNA2 Hs996788_m1 BIRC5 Hs _s1 CDC45 Hs97337_m1 E2F1 Hs153451_m1 E2F2 Hs231667_m1 MAX Hs81169_g1 GAPDH Hs _g1 Table S9. Primer used in this study Primer Location Primer Name Primer Sequence MYC_-2.2kb MYC_-2.2kbF ATGTTCATTAGCAGTGGTGATAGGTTA MYC_-2.2kbR AGGTGACTATTCAACCGCATAAGAG MYC_-1.6kb MYC_-1.6kbF GTGAGGGACCAAGGATGAGAAG MYC_-1.6kbR TTATTCCACGGCATGAAAAACA MYC_-1kb MYC_-1kbF TCTCCCGTCTAGCACCTTTGA MYC_-1kbR TTGCAACAGTCTCGGGCTG MYC_-.2kb MYC_-.2kbF ATTCATGCGGCTCTCTTACTCTG MYC_-.2kbR GGCAGCCGAGCACTCTAGC MYC_.1kb MYC_.1kbF GAGGGATCGCGCTGAGTATAAA MYC_.1kbR GCGAGTTAGATAAAGCCCCGA MYC_.8kb MYC_.8kbF CCCTTTCGAGATTTCTGCCTTAT MYC_.8kbR ATGTCCGACCGGCCG MYC_2.2kb MYC_2.2kbF ATGCCCCTCAACGTTAGCTTC MYC_2.2kbR ACCGAGTCGTAGTCGAGGTCAT MYC_3.7kb MYC_3.7kbF GCAACACCTGAAGTGTTCTTGGT MYC_3.7kbR AGCACATTCCCAAGCACCTC