Targeting P300 Addiction in CBP-Deficient Cancers Causes Synthetic Lethality by Apoptotic Cell Death Due to Abrogation of MYC Expression

Published OnlineFirst November 24, 2015; DOI: 10.1158/2159-8290.CD-15-0754

Research Article

Targeting p300 Addiction in CBP-Deficient Cancers Causes Synthetic Lethality by Apoptotic Cell Death due to Abrogation of MYC Expression

Hideaki Ogiwara1, Mariko Sasaki1, Takafumi Mitachi1, Takahiro Oike1,2, Saito Higuchi3, Yuichi Tominaga3, and Takashi Kohno1

Abstract Loss-of-function mutations in the CBP/CREBBP gene, which encodes a histone acetyltransferase (HAT), are present in a variety of human tumors, including lung, bladder, gastric, and hematopoietic cancers. Consequently, development of a molecular targeting method capable of specifically killingCBP- deficient cancer cells would greatly improve cancer therapy. Functional screening of synthetic-lethal genes in CBP-deficient cancers identified theCBP paralog

p300/EP300. Ablation of p300 in CBP-knockout and CBP-deficient cancer cells induced G1–S cell-cycle arrest, followed by apoptosis. Genome-wide gene expression analysis revealed that MYC is a major factor responsible for the synthetic lethality. Indeed, p300 ablation in CBP-deficient cells caused downregulation of MYC expression via reduction of histone acetylation in its promoter, and this lethality was rescued by exogenous MYC expression. The p300-HAT inhibitor C646 specifically suppressed the growth of CBP-deficient lung and hematopoietic cancer cellsin vitro and in vivo; thus p300 is a promising therapeutic target for treatment of CBP-deficient cancers.

SIGNIFICANCE: Targeting synthetic-lethal partners of genes mutated in cancer holds great promise for treating patients without activating driver gene alterations. Here, we propose a “synthetic lethal–based therapeutic strategy” for CBP-deficient cancers by inhibition of the p300 HAT activity. Patients with CBP-deficient cancers could benefit from therapy using p300-HAT inhibitors. Cancer Discov; 6(4); 1–16. ©2015 AACR.

See related commentary by Kadoch, p. xxx.

INTRODUCTION components of the SWI/SNF chromatin remodeling complex, such as SMARCA4/BRG1 and ARID1A/BAF250A, are Current approaches for precision medicine for treatment of frequently inactivated by protein truncation mutations and human cancer depend largely on targeting activated protein gross gene deletions (14, 16, 17). Notably, such chromatin kinases using specific inhibitors or antibodies. Kinase activation modifier genes work with and complement their structural in cancer is caused by genetic aberrations, such as the ABL gene and/or functional paralogs; consequently, cancer cells with fusion in chronic myelogenous leukemia, the ERBB2/HER2 deficiencies in chromatin modifiers are predicted to exhibit amplification in breast and gastric cancers, andEGFR muta- addiction (analogous to oncogene addiction) to the remain- tions and the ALK fusion in non–small cell lung carcinoma (1, ing intact paralogs. Based on this fact, we recently proposed a 2). Such genetic aberrations constitute a specific vulnerability of novel therapeutic strategy, “paralog targeting,” for use against cancer cells due to “oncogene addiction,” a state in which inhi- cancer cells with such gene deficiencies (18, 19). In this bition of the aberrant proteins causes death or growth arrest of approach, the remaining intact paralog of a deficient chroma- cancer cells. Recent studies by our group and others identified tin modifier gene is the target of therapeutic inhibition. We other kinase gene aberrations as promising therapeutic targets: first demonstrated the feasibility of this strategy by achieving FGFR family gene amplifications and fusions (3–6),AKT family specific killing of SMARCA4/BRG1-deficient cancers through gene mutations and fusions (7, 8), and fusions of RET, ROS1, inhibition of the SMARCA4 paralog SMARCA2/BRM-ATPase and NTRK1 (9–13). However, these studies also revealed that (19). These findings were subsequently supported by several only a small fraction of cancers carry such druggable aberra- studies, including some that employed genome-wide RNA tions in kinase genes; therefore, to further advance precision interference scanning (20–22). Recently, this concept has been cancer medicine, it is essential to identify non-kinase gene aber- extended to ARID1A-deficient cancers (23). Based on success- rations that can be targeted in the clinic. ful precision medicine approaches that have target-activated The most commonly mutated genes in human cancers oncogene products, paralog targeting strategies must meet encode chromatin modifiers (14, 15). Genes that encode the following criteria to be successfully translated to the clinic. First, cancer cells with a gene deficiency must exhibit 1Division of Genome Biology, National Cancer Center Research Institute, greater reliance on the remaining paralogs than cells with- 2 Tokyo, Japan. Department of Radiation Oncology, Gunma University Grad- out that deficiency, including noncancerous cells. Second, uate School of Medicine, Gunma, Japan. 3Oncology Research Laboratories, Daiichi-Sankyo Co., Ltd., Tokyo, Japan. the molecular mechanism underlying the addiction must be Note: Supplementary data for this article are available at Cancer Discovery proven. Third, existing inhibitors of the remaining paralog Online (http://cancerdiscovery.aacrjournals.org/). must exert a specific therapeutic effect on cancer cells harbor- Corresponding Author: Takashi Kohno, National Cancer Center Research ing a deficiency in a chromatin modifier. Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan. Phone: CBP/CREBBP (CREB-binding protein) and p300/EP300 81-3-3547-5272; Fax: 81-3-3542-0807; E-mail: [email protected] are chromatin modifier proteins that acetylate two lysine (K) doi: 10.1158/2159-8290.CD-15-0754 residues on histone H3, K18 and K27 (24, 25). CBP- and ©2015 American Association for Cancer Research. p300-mediated histone acetylation at gene promoter/enhancer

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RESEARCH ARTICLE Ogiwara et al. regions collaborates with SWI/SNF complexes to facilitate in the context of CBP deficiency indicates that p300 protein remodeling of chromatin into a relaxed state (26–28), allowing can replace some essential functions of CBP in CBP-deficient access by RNA polymerase II (29). We showed that approxi- cancer cells. This relationship is predicted to create a specific mately 10% to 15% of non–small cell and small cell lung cancers vulnerability, i.e., more severe functional loss of CBP should harbor loss-of-function aberrations in the CBP gene (30, 31). lead to a more pronounced dependency on p300. To examine Recent genome-wide sequencing studies reveal that such aber- the relationship between CBP and p300 in the same genetic rations are also prevalent in multiple types of human cancer, background, we established p300-KO cells in the H1299 including lymphoma (29%–33%), leukemia (18%), and bladder lung cancer cell line, as well as in the CBP-KO cells used cancer (15%–27%; refs. 22, 32–37). In addition, the recent detec- above (Supplementary Fig. S1E). Indeed, siRNA-mediated tion in lung cancer of deleterious mutations of KAT6B, which p300-knockdown (KD) caused marked suppression of growth encodes another histone acetyltransferase (HAT), expands the and survival in CBP-KO cells, but had little or no impact in known role of HAT gene aberrations in carcinogenesis (38). CBP WT cells (Fig. 1C and D and Supplementary Fig. S1F Recurrent missense mutations in CBP tend to cluster around and S1G). Similarly, CBP-KD caused marked suppression of the region encoding the HAT domain. In particular, mutations growth and survival in p300-KO cells, but not in p300 WT affecting the amino-acid residues p.Gly1411, p.Trp1472, and cells (Fig. 1C and E and Supplementary Fig. S1H and S1I). p.His1487, which ablate HAT and/or transcriptional coacti- Next, we investigated the impact on the cell cycle and apop- vation activity (30, 35), are frequently observed. In addition, totic cell death. In CBP-KO cells, depletion of p300 initially gross deletions and protein-truncating mutations are often caused G1 arrest, and then gradually induced apoptotic cell detected (6, 30, 35, 39). Development of therapeutic strate- death (Fig. 1F–H). Similarly, depletion of CBP in p300-KO gies for specific killing of CBP-deficient cancer cells will sig- cells caused G1 arrest and subsequent apoptotic cell death nificantly advance precision cancer medicine. In this study, a (Fig. 1F, I, J). Double depletion of CBP and p300 in cells profi- functional synthetic-lethal screen revealed that CBP-deficient cient for both genes caused transient growth suppression but cancer cells were killed by suppression of the paralog p300. did not affect sustainable colony formation; this observation

Here, we describe a paralog targeting strategy that exploits was further supported by the induction of transient G1 arrest, deficiency ofCBP in human cancers and satisfies the aforemen- but not apoptosis, under these conditions (Supplementary tioned criteria for successful translation into the clinic. Fig. S1J–S1N). These data indicated that lethality due to depletion of CBP and p300 is caused by apoptotic cell death, RESULTS and that this effect is specific to cells lacking thep300 and CBP genes, respectively. Identification of p300 as a Specific Synthetic- Lethal Gene in CBP-Deficient Cancer Cells Deregulation of MYC Transcription by p300-KD To identify synthetic-lethal partner genes in CBP-mutated in CBP-Deficient Cells cancer cells, we screened an siRNA library that targets genes Next, we performed genome-wide expression profiling involved in chromatin regulation, chromatin remodeling, his- analysis to define the genes underlying the synthetic-lethal tone modification, and histone marker recognition. To isolate relationship between CBP and p300. We identified 1,936 genes genes specifically required for growth ofCBP -mutated human whose expression levels changed >2-fold upon p300-KD in cancer cells, we initially performed the screen in two noncan- CBP-KO cells, and upon CBP-KD in p300-KO cells, but not cerous lung cell lines, HFL1 and MRC5; a CBP wild-type (WT) upon either KD alone in WT cells (Fig. 2A). Pathway analy- lung cancer cell line, A549; and a CBP-mutated (homozygous sis revealed that these genes were significantly P( < 0.001) deleted) lung cancer cell line, LK2. Six genes were identified enriched in 13 functional pathways, including those related as candidate lethal genes specifically inCBP -mutated cancer to G1–S cell-cycle control (ranks 1–4) and apoptosis (rank cells (Fig. 1A; Supplementary Table S1). We validated these 9), consistent with the phenotypes of G1 arrest and apop- six candidates using another set of human lung cancer cells: tosis observed following p300 depletion in CBP-KO cells or parental H1299 cells with intact CBP; H1299 cells with arti- CBP depletion in p300-KO cells (Fig. 2A and Supplementary ficial homozygousCBP -knockout (KO), designated H1299 Table S2). Notably, the MYC oncogene was the gene that 2G2; and a CBP-deficient lung cancer cell line, LK2. The top- appeared most frequently in these 13 functional pathways hit gene in this analysis was p300, whose knockdown exerted (Fig. 2A; Supplementary Fig. S2A and Supplementary Table antiproliferative and antisurvival effects specifically inCBP - S3). Quantitative RT-PCR confirmed that theMYC mRNA KO and CBP-deficient cells (Fig. 1B; Supplementary Fig. S1A– level was reduced upon p300 depletion in CBP-KO cells or S1C). No such effects of p300 depletion were observed in three upon CBP depletion in p300-KO cells (Fig. 2B). Consistent noncancerous cell lines: MRC5, HEK293T, and RPE1-hTERT with this, expression of MYC target genes, such as G1–S (Supplementary Fig. S1C and S1D). These data indicate that cyclins (Cyclin D1/CCND1 and Cyclin A2/CCNA2), cyclin- p300 is an essential factor specifically required for growth and dependent kinases (CDK4 and CDK6), and an antiapoptotic survival of CBP-mutated cancer cells. factor (Survivin/BIRC5), was also reduced in CBP-KO cancer cells (Supplementary Fig. S2B). Concordantly, levels of Synthetic Lethality of CBP and p300 Is the Result MYC proteins were specifically reduced inCBP -KO cells, but of G1 Arrest and Apoptosis not in CBP WT cells, upon p300 depletion (Fig. 2C). Cell CBP and p300 are paralogous HAT proteins with highly growth and MYC expression in CBP-KO cells in which endog- similar amino-acid sequences and some overlapping func- enous p300 was depleted by RNA interference targeting the tions (29, 40). Thus, identification ofp300 as a lethal gene 3′-untranslated region (UTR) of p300 was partially rescued by

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Targeting p300 Addiction in CBP-Deficient Cancers RESEARCH ARTICLE

A B 1st Screening 2nd Screening HFL1 MRC5 Cell growthColony formation 120 120 ) Cell lines H1299 LK2 Cell lines H1299 LK2 100 100 80 80 CBP status WT KO mu CBP status WT KO mu 60 60 siHDAC8 siHDAC8 40 Nonlethal genes 40 20 20 siDOT1L siDOT1L Proliferation (% Proliferation (%) Noncancerous in noncancerous Noncancerous 0 0 siRNA siRNA siSIRT4 siSIRT4 siSMARCA1 siSMARCA1 Nonlethal genes Candidate synthetic-lethal genes siKDM4B siKDM4B in CBP WT 6 Genes sip300 sip300 A549 LK2 120 120 ) ) 100 100 80 40 80 40 (% Proliferation (%) Survival (%) 80 Lethal genes 80 60 in CBPmut 60 40 Synthetic-lethal genes 40 in CBP-deficient cells 20 20 proliferation Proliferation (% CBP WT CBPmut 0 0 siRNA siRNA p300

C F siRNA siRNA Reseed Cell proliferation siRNA Cell cycle / Apoptosis

Day –2 –1 0 357 12 Day 0 2 58 Cell growth assay Colony formation assay FACS analysis

D Colony formation GHCell-cycle profile H1299 Apoptosis H1299 120 CBP-KO CBP-KO 80 ) 40 siNT siNT sip300 100 H1299 70 siNT siNT ) sip300 60 30 CBP WT sip300 sip300 (% 80

50 cells (% p300 WT 60 40 + 20 40 cycle (%) 30 CBP-KO Survival 20 10 20 p300 WT Cell 10 0 0 AnnexinV 0 CBP WT KO Day 258258258258 258 (Day) p300 WT WT G SG–M sub-G H1299 1 2 1

E IJ Colony formation Cell-cycle profile H1299 Apoptosis H1299

120 80 ) 40 H1299 siNT siCBP CBP-KO CBP-KO 100 siNT (%

) siNT siNT 60 30

(% 80 siCBP CBP WT sip300 sip300 cells

60 p300 WT 40 + 20 40 cycle (%) CBP WT Survival 20 10 20 p300-KO Cell 0 0 AnnexinV 0 p300 WT KO Day 258258258258 258 (Day) CBP WT WT H1299 G1 SG2–M sub-G1

Figure 1. Synthetic-lethal screening of chromatin modifier genes inCBP -deficient cancer cells. A, high-throughput screening to identify genes for which depletion is specifically lethal inCBP -deficient cancer cells. Noncancerous cells (HFL1 and MRC5),CBP WT cancer cells (A549), and CBP-deficient cancer (LK2) cells were seeded in 96-well plates and then transfected with siRNAs in duplicate. Each transfection plate contained 138 siRNAs targeting genes related to chromatin regulation; a pool of three different siRNAs targeted each gene. Cell viability was assessed 5 days after siRNA transfection. The screen identified six genes for which siRNA-mediated KD had little <( 35% inhibition) effect on cell viability in noncancerous cells and CBP WT cancer cells, but a marked (>65% inhibition) effect in CBP-deficient cells. B, identification of thep300 gene as a synthetic-lethal hit. H1299 (CBP WT), CBP-KO H1299 2G2 (CBP-KO), and CBP-deficient LK2 (CBP mu) cells were transfected with siRNAs for 48 hours, and then assayed for growth or colony formation. The relative proliferating ratio or surviving fraction of sip300-treated cells 5 days or 12 days after reseeding, respectively, is expressed as a heat-map plot of the percentage of H1299, CBP-KO H1299 2G2, and CBP-deficient LK2 cells transfected with targeting siRNAs that proliferated or survived, relative to the corresponding percentage in cells transfected with nontargeting siRNA (siNT). C–E, inhibition of cell growth by depletion of p300 or CBP. C, schematic time course of colony formation and cell growth assays. D and E, H1299 parental (CBP WT/p300 WT), H1299 CBP-KO (CBP-KO/p300 WT), or H1299 p300-KO (CBP WT/p300 KO) cells were transfected with siRNA (siNT, sip300 D1, or siCBP D2) for 48 hours, and then assayed for colony formation. The relative surviving fraction of siRNA-treated cells at 0 to 7 days or 12 days after reseeding, respectively, was expressed as the percentage of cells transfected with targeting siRNAs that survived, relative to the corresponding percentage in cells transfected with nontargeting siRNA. The num- bers and sizes of colonies formed by cells in which CBP and/or p300 were depleted are shown in the panels at right. Data, means ± SD. F–J, increase in the proportion of G1 phase and apoptotic cells upon depletion of CBP or p300. F, schematic time course of FACS analysis for examination of cell-cycle profile and apoptosis. G–J, cancer cells (G, H, H1299 CBP-KO 2G2; I, J, H1299 p300-KO #23) were transfected with siRNA (siNT, sip300 D1, or siCBP D2) for 48 hours. Two to eight days after reseeding, cell-cycle profiles and proportions of Annexin V–positive apoptotic cells were assessed by flow cytometry. Data, means ± SD.

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AB CBP WT p300 WT MYC mRNA MYC mRNA sip300/siNT: FC>±2 siCBP/siNT: FC>±2 1.2 1.2 75 1 1 45 29 0.8 0.8 283 105 49 0.6 0.6 0.4 0.4 179 72 siNT siNT 377 340 0.2 0.2 sip300 siCBP Relative expression Relative expression 0 0 117 1936 180 CBPWT KO p300 WT KO 3190 1953 H1299 H1299 p300 WT WT CBPWTWT

CBP-KO p300-KO sip300/siNT: FC>±2 siCBP/siNT: FC>±2 C H1299 H1299 H1299 CBP-KO p300-KO 13 Functional pathways including 1936 genes si p300 si CBP siNT si p300 si CBP p300 siNT siNT Overlapping Top 10 downregulated genes in a pathway Gene name p300 CBP pathways CBP 5 MYC MYC MYC MYC Down by sip300 KD Down by siCBP KD 4 CCND1 β-Actin β-Actin β-Actin 4 CDC45 Functional pathways Functional pathways 3 E2F2 D 2 E2F1 H1299 CBP-KO Cell growth g 2 CDT1 120 Vec p300HAT- 2 UBE2C 100

rankin 2 MCM2 80 2 MCM7 60 si p300 si p300 si p300 siNT siNT siNT p 10 2 MCM10 H1299 CBP-KO p300 40 To 2 BRCA1 siNT 20 2 CCNA2 MYC Proliferation (%) sip300_3′U MYC 2 ORC1L 0 β-Actin 2 TOP2A MYC commonly contributes to 5 pathways Vec 2 TYMS p300 HAT-

E Cell growth H1299 CBP-KO H1299 CBP-KO H1299 CBP-KO 120 Vec MYC Vec CCND1 Vec CDC45 100 80 60 H1299 CBP-KO si p300 si p300 si p300 si p300 si p300 si p300 siNT siNT siNT siNT siNT siNT 40 siNT p300 p300 p300 20

Proliferation (%) sip300 MYC CCND1 CDC45 0 Vec MYCCCND1 CDC45 β-Actin β-Actin β-Actin

Figure 2. MYC is a key determinant of cancer cell survival under p300- or CBP-depletion in CBP-KO or p300-KO cells, respectively. A, genome-wide gene expression analysis identifiedMYC as the gene most strongly associated with the observed synthetic lethality. A Benn analysis identified 1,936 genes that exhibited significant changes in expression specifically uponp300 depletion in H1299 CBP-KO cells and CBP depletion in H1299 p300-KO cells. WikiPathways database analysis identified the 13 top pathways P( < 0.001) comprising these 1,936 genes. MYC was identified as the gene most commonly included in the 13 pathways. B and C, expression profiles of H1299CBP/p300 WT and CBP-KO or p300-KO cells following p300 depletion. H1299 CBP/p300 WT, CBP-KO, and p300-KO cells were transfected with siNT, sip300 (D1), or siCBP (D2). After 48 hours, the cells were harvested and subjected to quantitative RT-PCR analysis and immunoblot analysis. The relative changes in gene expression relative to siNT are shown (B). Data, means ± SD. Immunoblot analysis of p300, CBP, MYC, and β-actin (loading control) (C). D, suppression of proliferation of H1299 CBP-KO cells by p300 depletion was rescued by exogenous expression of WT p300, but not HAT-defective p300. H1299 CBP-KO cells were transfected with a plasmid expressing p300 or HAT-defective p300 (HAT-) cDNAs. Twenty-four hours after transfection, the cells were further transfected with sip300 (3′UTR: targeting the 3′ UTR region of the p300 mRNA) or siNT. Forty-eight hours later (day 0), cells were seeded in 96-well plates. Cell viability was measured at 5 days. Immunoblot analyses of p300, MYC, and β-actin (loading control). Data, means ± SD. E, suppression of proliferation of H1299 CBP-KO cells by p300 depletion rescued by exogenous expression of MYC, but not CCND1 and CDC45. H1299 CBP-KO cells with or without stable exogenous MYC, CCND1, or CDC45 cDNA expression were transfected with siNT or sip300 (D1). Forty-eight hours later (day 0), cells were seeded in 96-well plates. Cell viability was measured at 5 days. Immunoblot analyses of p300, MYC, CCND1, CDC45, and β-actin (loading control) at 48 hours are shown. Data, means ± SD. Vec, vector.

exogenous expression of WT p300, but not a HAT-deficient tional pathways (Fig. 2A), did not rescue the suppression of mutant p300 (Fig. 2D; Supplementary Fig. S2C). Further- H1299 CBP-KO cell growth by p300 depletion (Fig. 2E). On more, cell growth suppression mediated by p300 depletion in the other hand, downregulation of these two genes and other H1299 CBP-KO cells was rescued by exogenous expression of top-ranked genes, E2F2 and E2F1, by p300 depletion in CBP- MYC (Fig. 2E; Supplementary Fig. S2D). Similar results were KO cells was rescued by exogenous MYC expression (Supple- also obtained in CBP-KO HEK293T cells (Supplementary Fig. mentary Fig. S2G), suggesting that MYC plays a key role in S2E and S2F), confirming the results in H1299 cells. the lethality of CBP-deficient cells by p300 depletion. MYC Exogenous expression of CCND1 and CDC45, the two genes protein is activated by dimerization with MAX protein (41). appearing most frequently (other than MYC) in the 13 func- In fact, suppression of H1299 CBP-KO cell growth by p300

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Targeting p300 Addiction in CBP-Deficient Cancers RESEARCH ARTICLE depletion was partially rescued by exogenous MAX expres- (Fig. 3C). Accordingly, recruitment of RNA polymerase II sion (Supplementary Fig. S2H), indicating that deficiency (RNAPII) to the TSS was markedly reduced upon p300 deple- of the MYC–MAX complex underlies the lethality of p300 tion in CBP-KO cells, and the phosphorylation of serine 5 of depletion in CBP-deficient cells. Taken together, these data RNAPII, which is a marker of readiness for transcriptional suggest that the vulnerability of CBP-KO cells is at least initiation, was more impaired by depletion of p300 in CBP- partially due to p300 HAT–dependent expression of MYC. KO cells than in CBP-proficient cells (Fig. 3C). Therefore, both p300 and CBP redundantly acetylate histones within the p300 Depletion–Mediated Dysregulation of MYC gene locus, allowing the transcription of MYC. However, Chromatin Modification Causes Downregulation in CBP-KO cells, MYC expression is largely dependent on of MYC histone acetylation by p300 and is consequently repressed by Next, we investigated the molecular mechanisms of MYC p300 depletion. transcriptional repression in CBP-deficient cells following p300 depletion. Chromatin immunoprecipitation (ChIP) Addiction to p300 Due to Deleterious CBP assays revealed that p300 and CBP localized to enhancer, Gene Aberrations promoter, and exon regions around the transcription start We next investigated whether the proliferation and sur- site (TSS) of the MYC gene in CBP-proficient, CBP-mutant, vival of other CBP-mutated cancer cell lines depends on and CBP-KO cells (Fig. 3A and B and Supplementary Fig. p300. siRNA-mediated p300 depletion impaired both the sur- S3). Acetylation at H3K18 and H3K27, which are redun- vival and proliferation of cancer cells carrying missense CBP dantly acetylated by p300 and CBP (24), was abundant in the mutations with highly deleterious affect HAT activity (H520, promoter region (Fig. 3C). p300 depletion in CBP-KO cells H1703, and TE10) or nonsense mutations, resulting in trun- decreased the occupancy of acetylated H3K18 and H3K27 in cation of the CBP protein (TE8 and LK2; Fig. 4A for survival; the promoter region relative to that in CBP-proficient cells Supplementary Fig. S4A and S4B for cell growth). However,

ABCBP and p300 ChIP 0.5 0.4 –2.2kb –1.6kb –1kb –0.2kb 0.1 kb 0.8 kb 2.2kb 3.7kb )

(% 0.3 IgG Enhancer Promoter MYC gene 0.2 Input CBP 0.1 p300 0 –2.5 –2 –1.5 –1 –0.5 00.5 1 1.5 2 2.5 3 3.5 4 Distance from TSS of MYC gene (kb) C H3K18ac ChIP H3K27ac ChIP 8 5 7 4

) 6 ) 5 (% CBP WT siNT (% 3 CBP WT siNT 4 3 CBP WT sip300 2 CBP WT sip300 Input Input 2 CBP-KO siNT 1 CBP-KO siNT 1 CBP-KO sip300 CBP-KO sip300 0 0 –2.5–2 –1.5–1 –0.50 0.51 1.5 22.5 3 3.5 4 –2.5 –2 –1.5 –1 –0.5 00.5 1 1.5 2 2.5 3 3.5 4 Distance from TSS of MYC gene (kb) Distance from TSS of MYC gene (kb)

RNAPII ChIP RNAPII pS5 ChIP 2.5 2.5 2 2 ) ) (% 1.5 CBP WT siNT (% 1.5 CBP WT siNT 1 CBP WT sip300 1 CBP WT sip300 Input Input 0.5 CBP-KO siNT 0.5 CBP-KO siNT CBP-KO sip300 CBP-KO sip300 0 0 –2.5–2 –1.5–1 –0.50 0.51 1.5 2 2.5 33.5 4 –2.5 –2 –1.5 –1 –0.5 0 0.5 1 1.5 22.5 3 3.5 4 Distance from TSS of MYC gene (kb) Distance from TSS of MYC gene (kb)

Figure 3. MYC transcriptional initiation is inactivated by suppression of acetylation of histone H3K18/K27 upon p300 depletion in CBP-deficient cancer cells. A, location of the PCR amplicons in the MYC locus used for the ChIP assay. Distances from the transcription start site (TSS) are indicated in kb. Quantitative genomic PCR was performed using input and ChIP DNAs. Relative ChIP enrichment values in regions at the indicated distances from the transcription start site in the MYC gene are expressed as percentages relative to input DNA. Data, means ± SD. B, p300 and CBP proteins localized upstream of the MYC locus. ChIP assays were performed on H1299 cells using control IgG or antibodies against CBP or p300. C, acetylation of histone H3 and activation of RNA polymerase II (RNAPII) in the MYC locus. ChIP assays were performed on parental H1299 and CBP-KO H1299 cells to examine acetylation of histone H3K18 and H3K27 and the recruitment of RNAPII and phosphorylation of RNAPIIS5 48 hours after transfection with siNT or sip300 D1.

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A CBP status p300 status siNT Survival (%) sip300 Functional Functional Mutation impact or Mutation impact or 020406080 100 120 Cell lines deletion deletion A549 WT – WT – H1299 WT – WT – H661 WT – WT – H157 WT – WT – CBP proficient SQ5 WT – WT – SW480 WT – WT – SW620 WT – P1440L Medium HT29 WT – M1470fs Del HCT116 WT – K1648fs Del Lovo T573 Low WT – H322 S893L Low WT – CBP low/medium H2009 H67R Low WT – impact mutation H1048 S1680del Medium WT – H2087 I1846M Medium D1485splice Del KM12C R1664C Medium P1452L/ Q761R High/Medium H520 R1446C High WT – CBP deleterious or TE8 Q1765fs Del WT – high-impact LK2 HD ex3 Del WT – mutation H1703 W1472C/N2175S High/Low A1437V Medium TE10 Y1539C High W1509C High

BCCell-cycle profile Apoptosis D 70 4 siNT siNT 60 CBP WT CBPmut ) of 50 sip300 3 sip300

cells H157 SQ5 LK2 H1703 40 + 2 change cycle (% 30 ld

20 si p300 si p300 si p300 si p300 1 siNT siNT siNT siNT Cell Fo 10 AnnexinV p300 0 0 1 1 1 1 1 1 1 1 5 2 3 S S S S MYC G G G G G G G G –M –M –M –M LK 2 2 2 2 SQ

H157 β-Actin G G G G H170 Sub- Sub- Sub- Sub-

H157 SQ5LK2 H1703 CBP CBP WT mut CBP WT CBPmut

Pro TSS E F EP300 mRNA MYC mRNA –1kb 0.1 kb 1.2 n siNT Enhancer Promoter MYC gene 1 sip300 0.8 p300 ChIP H3K27ac ChIP RNAPII ChIP 0.6 0.15 6 0.8 IgG siNT 0.7 siNT 0.4 5 p300 sip300 0.6 sip300 ) ) ) 0.2 0.1 4 0.5 (% (% (% Relative expressio 0 3 0.4 5 2 3 5 2 3 0.3 Input Input

Input 0.05 2 LK LK SQ SQ 0.2 H157 H157 H170 H170 1 0.1 0 0 0 CBP CBP CBP CBP ProTSS ProTSS ProTSS WT mut WT mut MYC gene region MYC gene region MYC gene region

GHH1299 shp300 LK2 shp300 LK2 shp300 I LK2 xenograft ) ) ) 3,000 1.4 2,500 1,500 3 – 3 3 Dox– – Dox Dox m m m 2,500 + 1.2 * 2,000 + + Dox Dox Dox weight 1 1,000 2,000 1,500 0.8 1,500 1,000 0.6 1,000 * 500 0.4 – 500 * Dox

mor volume (m 500 mor volume (m mor volume (m * 0.2 Dox+

* Tu Relative tumor Tu Tu 0 0 * 0 0 0102030 0102030 0102030 shNT shp300 Day Day Day

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Targeting p300 Addiction in CBP-Deficient Cancers RESEARCH ARTICLE p300 depletion had no apparent effect on cancer cells carry- (shNT) or p300-targeting (shp300) shRNAs were injected into ing missense CBP mutations with low or medium impacts on the flanks of immune-deficient mice. When mice were fed the HAT domain (LoVo, H322, H2009, H1048, H2087, and doxycycline immediately after injection to induce p300 deple- KM12C), or on CBP WT cells, regardless of p300 mutation tion, growth of LK2-shp300 xenografts was significantly sup- (Fig. 4A for survival; Supplementary Fig. S4A and S4B for cell pressed, whereas the growth of LK2-shNT, H1299-shNT, and growth). Similar results were also obtained by depleting p300 H1299-shp300 xenografts was unaffected (Fig. 4G; Supple- with siRNAs targeting different sites, and also by constitutive mentary Fig. S4L). When mice with engrafted tumors derived expression of a lentivirally encoded shRNA (Supplementary from CBP-deficient LK2 lung cancer cells were fed doxycy- Fig. S4C–S4E). Taken together, these data demonstrate that cline to induce p300 depletion, the tumor growth and weight cancer cells that are CBP deficient due to loss-of-function of LK2-shp300 xenografts, but not LK2-shNT xenografts, was mutations or deleterious aberrations are addicted to p300. significantly impaired (Fig. 4H and I and Supplementary Fig. As in the case of the CBP-KO cells described above, the S4M). Growth suppression of LK2 cells by p300 depletion apoptotic fraction specifically increased inCBP -deficient can- was also observed in a mouse orthotopic model, in which cer cells upon p300 depletion (Fig. 4B and C). These CBP- lung cancer cells were implanted into the thoracic cavities of deficient cancer cells underwent apoptosis associated with mice (Supplementary Fig. S4N). These data demonstrate the PARP cleavage, but did not exhibit senescence or autophagy in vivo therapeutic potential of p300 inhibition against CBP- (Supplementary Fig. S4F). Expression of MYC mRNA and deficient lung cancers. MYC protein was specifically reduced by p300 depletion in CBP-deficient cancer cells (Fig. 4D and E). Growth suppres- Sensitivity of CBP-Deficient Lung Cancer Cell to sion by p300 depletion in CBP-deficient LK2 cancer cells was an Existing p300 HAT Inhibitor partially rescued by exogenous MYC expression (Supplemen- To determine whether inhibition of p300 HAT activity is tary Fig. S4G). In addition, in CBP-deficient cancer cells, p300 viable as a therapeutic strategy against cancer, we examined preferentially localized to the promoter region of the MYC the ability of an existing specific inhibitor of p300 HAT, C646 gene, and p300 depletion impaired histone acetylation and (42), to suppress the growth of CBP-deficient lung cancer RNAPII localization in that region (Fig. 4F). Therefore, in cells. C646 reduced survival in lung cancer cells carrying del- CBP-deficient cancer cells, p300-dependentMYC expression eterious CBP mutations (H1703, H520, and LK2) to a greater is essential for cell survival. extent than in CBP WT cells (A549, H1299, and H157) or cells harboring low-impact mutations (H322; Fig. 5A and Supple- Therapeutic Potential of p300 Inhibition against mentary Fig. S5A). As in the case of p300-KD, C646 treatment CBP-Deficient Cancers led to a marked increase in the apoptotic fraction specifically Next, using a mouse xenograft model, we investigated the in CBP-deficient cancer cells (Fig. 5B and Supplementary Fig. in vivo therapeutic potential of p300 inhibition against CBP- S5B). Accordingly, the level of MYC expression was specifi- deficient cancers. For these experiments, we establishedCBP cally reduced in CBP-deficient cells (Fig. 5C and D and Sup- WT H1299 and CBP-deficient LK2 lung cancer cells in which plementary Fig. S5C and S5D). In addition, in CBP-deficient expression of p300 could be conditionally knocked down cancer cells, localization of acetylated histone H3 and RNAPII with doxycycline (Supplementary Fig. S4H). shRNA-mediated in the promoter region of the MYC locus was impaired follow- p300 depletion in CBP-deficient LK2 lung cancer cells, but ing treatment with C646, concomitant with a slight decrease not in CBP WT H1299 cancer cells, led to a reduction of in global acetylation levels of H3K18 and H3K27 (Fig. 5D and growth and survival and an increase in the rate of apoptosis Supplementary Fig. S5D and S5E). Finally, we examined the in vitro (Supplementary Fig. S4I–S4K). H1299 and LK2 lung therapeutic potential of C646 in a mouse xenograft model. cancer cells carrying conditionally expressed nontargeting C646 treatment led to a significant reduction in the growth

Figure 4. p300 depletion is lethal in cancer cells harboring loss-of-function mutations in CBP. A, synthetic-lethal effects assessed by colony formation assay. Data regarding missense and deleterious (Del) mutations were obtained from the cBioPortal and COSMIC databases. Functional impact values (neutral, low, medium, and high) of missense mutations were obtained from the MutationAssessor database. Cells were transfected with siRNA (siNT or sip300 Dp) for 48 hours and then assayed for colony formation. The surviving fraction of sip300-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. Data, means ± SD. B–E, increase in the proportions of G1 phase and apoptotic cells following depletion of p300 in CBP-deficient cancer cell lines. H157 and SQ5 (CBP WT) cells and H1703 and LK2 (CBP-deficient) cells were transfected with siRNA (siNT, sip300 Dp) for 48 hours; 96 hours after reseeding, the cells were subjected to flow cytometry to determine the cell-cycle profileB ( ) and the proportion of Annexin V–positive apoptotic cells (C). Immunoblot analysis (D) and quantitative RT-PCR analysis (E) 48 hours after siRNA-mediated p300 depletion of CBP WT and CBP-mutant cancer cells. Immunoblot analysis was performed using antibodies against p300, MYC, and β-actin (loading control). Expression levels of p300 and MYC were normalized against the level of GAPDH mRNA in the same samples. Data, means ± SD. F, suppression of transcriptional initiation of the MYC gene by impairment of histone acetylation upon p300 depletion in CBP-deficient LK2 lung cancer cells. Locations of the PCR amplicons in the MYC locus used for the ChIP assay (upper panel). The transcription start site (TSS) and promoter region (Pro) are indicated. Localization of p300 proteins at TSS and Pro in the MYC locus (left panel). Acetylation of Histone H3K27 (middle) and localization of RNA polymerase II (RNAPII; right) in the MYC locus 48 hours after transfection with siNT or sip300 Dp. G, suppression of in vivo growth of tumor cells by p300 depletion immediately after injection. H1299-shp300 and LK2-shp300 cells were implanted subcutaneously into BALB/c-nu/nu mice, and the mice were randomly divided into two groups: one group was fed a diet containing doxycycline (Dox+), and the other was fed a control diet (Dox−). Tumor volumes for each group are shown. H and I, suppression of in vivo growth of tumor cells by p300 depletion allowed to engraft after injection. LK2-shp300 cells were implanted subcutaneously into BALB/c-nu/nu mice. When the tumors reached more than 200 mm3, the mice were randomly divided into two groups and fed a diet containing doxycycline (Dox+) or a control diet (Dox−). Changes in tumor volume in both groups (H). Tumor weights at the time of sacrifice in both groups (I). Asterisks indicate significant differences in tumor volume or weight between doxycycline-fed and control mice (P < 0.05; Student t test). Data, means ± SE.

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B A Survival (%) Apoptosis 10 – Cell lines 1 10 100 ) C646 H661 8 C646+

A549 cells (% 6

CBP proficient + H1299 4 H157 2

CBP low-impact mutation H322 AnnexinV 0 H520 CBP deleterious or H157 LK2 high-impact LK2 C646– mutation H1703 C646+ CBP CBP WT mut

CDH157 LK2 E MYC mRNA C646 –+ –+ LK2 xenograft 1.2 1,400 MYC )

3 Mock n

1 m 1,200 β-actin C646 0.8 1,000 0 µmol/L H157 LK2 800 0.6 15 µmol/L C646 –+ –+ 600 0.4 * 20 µmol/L H3 400

mor volume (m * 0.2 200 * * Relative expressio

30 µmol/L H3K18ac Tu 0 10.9710.87 0 010203040 H157 LK2 H3K27ac 11.0510.80 Day CBP CBP WT mut

Figure 5. Response of CBP-deficient lung cancer cells to the p300 HAT inhibitor C646. A, survival of CBP-deficient cancer cells after treatment with C646. Cells were subjected to a colony formation assay in the presence or absence of 15 μmol/L C646. Surviving C646-treated cells are shown as a percentage of nontreated cells. Data, means ± SD. B, induction of apoptosis by C646 treatment. CBP WT H157 and CBP-deficient LK2 cells were treated with C646 [0 μmol/L (C646−) or 15 μmol/L (C646+)] for 48 hours, and then the proportion of Annexin V–positive apoptotic cells was assayed by flow cytometry. Data, means ± SD. C, expression of the MYC gene in cells treated with C646. CBP WT H157 and CBP-deficient LK2 cells were subjected to quantitative RT-PCR analysis 48 hours after treatment with the indicated concentrations of C646. The fold-change in expression of MYC is expressed relative to the level in nontreated cells. D, MYC protein expression in cells treated with C646. CBP-deficient LK2 and WT H157 cell lines were treated with C646 [0 μmol/L (C646−) or 15 μmol/L (C646+)] for 48 hours, harvested, and subjected to immunoblot analysis with antibodies against MYC, acetylated histone H3K18 (H3K18ac), H3K27 (H3K27ac), and β-actin (loading control). The ratios of the levels of acetylated histone H3K18 and H3K27 levels (normalized to total H3 levels) to the corresponding levels in C646-untreated cells are shown below. E, effect of C646 on tumor growth in vivo. CBP- deficient LK2 cells were implanted subcutaneously into BALB/c-nu/nu mice. Twenty days after implantation, the mice were randomly divided into two groups and intraperitoneally injected with C646 (25 mg/kg) or vehicle alone once a day for 14 days (arrows). Asterisks indicate significant differences in tumor volume between the C646-treated and mock-treated mice (P < 0.05; Student t test). Data, means ± SD. of CBP-deficient LK2 lung cancer cells (Fig. 5E), without any suggest that growth suppression of CBP-deficient lung cancer significant loss in body weight (Supplementary Fig. S5F). cells can be achieved by specific inhibition of p300 activity by Thus, both in vitro and in vivo growth of CBP-deficient lung targeting its bromodomain as well as its HAT activity. cancer cells depends on p300 HAT activity. C646 also exhibited therapeutic potential in the deadliest type of lung cancer, CBP-Deficient Hematopoietic Cancer Cells small cell lung cancer (SCLC), which frequently harbors CBP Depend on p300 Function deficiency (ref. 31; Supplementary Fig. S5G and S5H). Thus, The CBP gene is frequently mutated in hematopoietic can- CBP-deficient lung cancers are vulnerable to p300 inhibition cers (22, 32–37). Notably, among cancers deposited in the Inter- irrespective of histologic subtypes. national Cancer Genome Consortium database (https://icgc. We next investigated the therapeutic potential of other org/), malignant lymphoma exhibits the highest rate of CBP compounds that inhibit p300 activity. L002, a multiple HAT mutation (18.2%; Supplementary Fig. S6A). Hence, we exam- inhibitor that targets not only p300 but also other HATs such ined the effects of C646 on the growth of hematopoietic cancer as CBP, PCAF, and GCN5 (43), did not exert specific toxicity cells, including malignant lymphoma, carrying CBP mutations according to the CBP status of lung cancer cell lines (Supple- (33–35). As observed in lung cancer cells, C646 treatment mentary Fig. S5I). On the other hand, lung cancer cells carrying markedly reduced the proliferation of cancer cells harboring deleterious CBP mutations were sensitive to two bromodomain a loss-of-function mutation in the HAT domain (WSU-NHL) inhibitors targeting p300 and CBP, SGC-CBP30 and I-CBP112 or deleterious mutations in the CBP gene (SU-DHL-6, VAL, (refs. 44, 45; Supplementary Fig. S5I). These observations Jurkat, and SU-DHL-5; Fig. 6A and Supplementary Fig. S6B).

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Targeting p300 Addiction in CBP-Deficient Cancers RESEARCH ARTICLE

A CBP status p300 status C646– Functional Proliferation (%) C646+ Functional impact impact or Mutation or deletion Mutation 020406080100 120 deletion Cell lines U2932 WT – WT – Loucy WT – WT – CBP proficient RC-K8 WT – DelC-1087 Del RL WT – E1011X/H1415PDel/Medium CBP low-impact mutation Farage Q1079H Low M1470fsDel WSU-NHL H1487Y High WT –

CBP deleterious or VAL Q232XDel WT – high-impact Jurkat F1358fs/R1563S/P2311T Del/Medium/LowWT– mutation SUDHL5 1 copy Del (LOH) Del WT – SUDHL6 L470fsDel R1627W High

Jurkat BCCell-cycle profile Apoptosis D C646 (µmol/L) 0510 100 100 ) MYC 80 (% 80 β-actin

60 cells 60 clevPARP

cycle (%) 40 0 µmol/L 40 0 µmol/L Jurkat 5 µmol/L 5 µmol/L C646 (µmol/L) 0510 Cell 20 20

10 µmol/L AnnexinV 10 µmol/L H3 0 0 61648616 48 61648616 48 (hour) 61648 (hour) H3K18ac 10.890.77 H3K27ac G1 SG2–M sub-G1 10.870.46 Cell growth EF150 Day 4 Day 7 Day 9 Day 11 Jurkat shNT shp300 shNT shp300 shNT shp300 shNT shp300 100 Dox –+–+ –+ –+ –+–+ –+–+ Day 4 p300 Day 7 50 MYC Day 9 Proliferation (%) Day 11 β-actin 0 H3K18ac (Dox) –+–+ clevPARP shNT shp300

GHCell-cycle profile shNT Dox– Apoptosis 60 40 shNT Dox+ 50 –

shp300 Dox ) 40 30

+ (% shp300 Dox 30 + – cycle (%) 20 shNT Dox 20 +

Cell shNT Dox 10 AnnexinV 10 shp300 Dox– 0 shp300 Dox+ 479114 7911 47911 47911 (Day) 0 47911 (Day) G1 S G2–M sub-G1

IKJurkat shp300 J Jurkat xenograft Jurkat xenograft – ) ) 4,000 Dox 250

1.4 3 3 Mock m m + 1.2 * 3,000 Dox 200 C646 weight 1 150 2,000 0.8 0.6 100 1,000 0.4 – Dox 50 mor volume (m mor volume (m * * 0.2 + * * * * Tu Tu Dox 0 ** *** *** Relative tumor 0 0 010203040 (Day) shNT shp300 0510 15 20 25 30 35 40 45 (Day)

Figure 6. Depletion or inhibition of p300 suppresses growth of hematopoietic cancers with loss-of-function mutations in CBP. A, human hematopoietic cancer cell lines used to examine sensitivity to C646. Data regarding missense and deleterious (Del) mutations were obtained from the cBioPortal and COSMIC databases. Functional impact values (neutral, low, medium, and high) of missense mutations were obtained from the MutationAssessor database. Proliferation of hematopoietic cancer cell lines was examined 5 days after treatment with or without 10 μmol/L C646. Proliferation of C646-treated cells is expressed as a percentage of proliferation in nontreated cells. Data, means ± SD. B–D, induction of apoptosis by C646 treatment. Jurkat (CBP-deficient) cells were treated with C646 (0, 10, or 15 μmol/L) for 6, 16, or 48 hours, and then assayed by flow cytometry to determine the cell-cycle profileB ( ) and the proportion of Annexin V–positive apoptotic cells (C). Data, means ± SD. Immunoblot analysis (D) was performed using antibodies against MYC, histone H3 AceK18 (H3K18ac), histone H3 AceK27 (H3K27ac), cleaved PARP, and β-actin (loading control). The ratios of the levels of acetylated histone H3K18 and H3K27 levels (normalized to total H3 levels) to the corresponding levels in C646-untreated cells are shown below. E and F, p300 depletion causes lethality of CBP-deficient hematopoietic cancer cells. Jurkat-shNT and Jurkat-shp300 cells were cultured in the presence or absence of doxycycline (Dox) before analysis. E, immunoblot analysis. F, cell proliferation assessed by cell-counting assay. Data, means ± SD. G and H, Induction of apoptosis by depletion of p300. Jurkat-shNT and Jurkat-shp300 cells were cultured in the presence or absence of doxycycline. After harvest, cell-cycle profiles and the proportions of Annexin V–positive apoptotic cells were assayed by flow cytometry. Data, means± SD. I and J, suppression of tumor growth in vivo by p300 depletion. Jurkat-shp300 cells were implanted subcutaneously into BALB/c-nu/nu mice. When the tumors reached more than 200 mm3, mice were randomly divided into two groups and fed either a diet containing doxycycline (Dox+) or a control diet (Dox−). Changes in tumor volume in both groups are shown (I). Tumor weights at the time of sacrifice in both groups J( ). K, suppression of tumor growth in vivo by a p300-HAT inhibitor C646. CBP-deficient Jurkat cells were implanted subcutaneously into BALB/c-nu/nu mice, and the growth of xenografts was examined. Twenty days after implantation, the mice were randomly divided into two groups and intraperitoneally injected with C646 (25 mg/kg) or vehicle alone once a day for 14 days (arrows). Asterisks indicate significant differences in tumor volume between doxycycline-fed and control mice P( < 0.05; Student t test). Data, means ± SD.

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These results strongly indicate that the growth of CBP-deficient Jurkat cells carrying conditionally expressed nontargeting hematopoietic cancer cells can also be suppressed by drugs that (shNT) or p300-targeting (shp300) shRNAs into the flanks of inhibit the HAT activity of p300 protein. On the other hand, immune-deficient mice. When mice with engrafted tumors CBP-deficient hematopoietic cancer cells were not specifically derived from CBP-deficient Jurkat cells were fed doxycy- sensitive to the p300/CBP bromodomain inhibitors described cline to induce p300 depletion, the growth of Jurkat-shp300 above (Supplementary Fig. S6C). This might be due to the fact xenografts, but not Jurkat-shNT xenografts, was significantly that bromodomain inhibitors have multiple targets, such as impaired (Fig. 6I and J and Supplementary Fig. S6F). Finally, BRD4, which is therapeutically targeted in hematopoietic can- we examined the therapeutic potential of C646 in a mouse cer to downregulate MYC (44). xenograft model. C646 treatment significantly decreased the C646 treatment led to an increase in the proportion of growth of CBP-deficient Jurkat cancer cells (Fig. 6K). Thus, apoptotic cells in CBP-deficient hematopoietic cancer cells both in vitro and in vivo growth of CBP-deficient hemato (Fig. 6B and C). Similarly, C646 treatment also led to the poietic cancer cells depends on p300-HAT activity, further induction of apoptosis in other CBP-deficient, but notCBP confirming the feasibility of p300 targeting as a method for WT, cancer cells (Supplementary Fig. S6D). C646 treatment treatment of CBP-deficient cancers. of CBP-deficient cancer cells also reduced the levels of MYC protein, slightly decreased global histone H3 acetylation, and promoted the cleavage of PARP, a marker of apoptosis DISCUSSION (Fig. 6D). To further examine the effect of p300 inhibition, Here, we identified p300 HAT as a promising candidate for we established CBP-deficient hematopoietic Jurkat cells in a paralog targeting strategy for treating cancers that harbor which p300 expression could be conditionally knocked down deleterious aberrations of the chromatin modifier CBP. p300 by the addition of doxycycline. Consistent with the results inhibition in CBP-deficient cancer cells caused downregula- of C646 treatment, shRNA-mediated p300 depletion led to a tion of MYC and specifically led to the death of CBP-deficient reduction in cell growth, concomitant with an increase in the cancer cells; thus, the targeting of p300 holds great promise proportion of apoptotic cells (Fig. 6E–H and Supplementary for the treatment of CBP-deficient cancers (Fig. 7). The fre- Fig. S6E). In addition, as in the case of C646 treatment, the quency and prevalence of mutations that inactivate chroma- levels of MYC protein, acetylated histone H3, and PARP cleav- tin modifiers in a wide range of human cancers indicate that age were also increased by doxycycline-induced p300 deple- such mutations are driver alterations that cause loss of tumor- tion (Fig. 6E). These findings suggest that p300 inhibition suppressive functions (14). The promise of paralog targeting represents a viable therapeutic strategy against CBP-deficient in precision cancer medicine has been further validated by hematopoietic cancers. the addition of the strategy described here to the previ- We next investigated the in vivo therapeutic potential of ously established repertoire, e.g., the use of SMARCA2/BRM- p300 inhibition against CBP-deficient hematopoietic cancers ATPase inhibition against SMARCA4/BRG1-deficient cancers using a mouse xenograft model. To this end, we injected and ARID1B inhibition against ARID1A-deficient cancers

Patient with CBP-deficient cancer Normal cells CBP-deficient cancer cells

CBP Paralog p300 CBP Paralog p300 MYC MYC CBP/p300 p300 redundancy addiction Survival Survival

p300 targeting therapy

HAT Inhibitor HAT Inhibitor

CBP p300 CBP p300 MYC MYC MYC MYC Cell death proficiency deficiency Survival Survival Low Synthetic Apoptosis impact lethality

Low side effects High therapeutic effects

Figure 7. Targeting p300 in CBP-deficient cancers causes synthetic lethality via apoptotic cell death due to impairment ofMYC expression.

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Targeting p300 Addiction in CBP-Deficient Cancers RESEARCH ARTICLE

(19–23). Thus, the paralog targeting strategy should be appli- Moreover, p300 depletion in CBP-deficient cancer cells did cable to a considerable fraction of human cancers. not significantly affect the accumulation of BRD4 at theMYC We revealed the synthetic-lethal relationship between CBP promoter (Supplementary Fig. S7), and CBP-deficient Jurkat and p300 using human cancer cells whose CBP or p300 genes cells are not susceptible to a BRD inhibitor (50, 54); therefore, were artificially knocked out by genome editing. Cancer cells the vulnerability of CBP-deficient cancer cells to p300 inhibi- with inactivating CBP mutations (i.e., deleterious mutations tion is likely to be achieved in a BRD4-independent manner. with high functional impact) and WT p300 were evidently Our results revealed the therapeutic utility of the exist- more susceptible to p300 suppression than cells with WT ing p300 HAT inhibitor C646 (42) for personalized cancer or minimally compromised CBP, indicating that cancer cells therapy based on specific genetic alterations.CBP -mutant with CBP deficiency have become addicted to p300 activity. lung and hematopoietic cancer cells were more sensitive than On the other hand, siRNA-mediated transient double deple- WT cells to C646, reflecting a cellular context vulnerable to tion of CBP and p300 in cells proficient for both genes did p300 inhibition. In particular, cancer cells carrying gross dele- not cause evident lethality. This result is consistent with tions, protein-truncating (nonsense and frameshift) muta- a previous finding that conditional knockout of both the tions, or missense mutations in the HAT domain, as well as Cbp and p300 genes in mice leads to cell-cycle arrest, but not other predicted loss-of-function mutations, exhibited higher apoptosis (24). The profound addiction to p300 inhibition sensitivity to C646 than those harboring low- or medium- exhibited by CBP-deficient cancer cells is critical for transla- impact missense mutations in CBP. Therefore, patients with tion of p300-inhibitory therapy to the clinic. cancers carrying such deleterious CBP aberrations should Our findings indicate thatMYC plays a key role in deter- benefit from therapy with p300 HAT inhibitors.CBP muta- mining the survival of CBP-deficient cancer cells in the con- tions in cancer cells are often heterozygous (leaving one text of p300 depletion. Expression of MYC in CBP-deficient CBP allele intact), indicating that haploinsufficiency of CBP cancer cells was downregulated following either RNA inter- or dominant-negative activities of mutant CBP can drive ference– or HAT inhibitor–mediated inhibition of p300. tumorigenesis (6, 30). In fact, cancer cell lines such as H1703, This finding is reasonable in light of several previous stud- H520, Jurkat, VAL, and WSU-NHL, which were sensitive to ies showing that MYC upregulates the expression of cell p300 inhibition, harbor heterozygous CBP mutations. CBP growth–related and antiapoptotic factors that interact with protein was recruited to the MYC promoter region in can- CBP and p300 (46–49). Our genome-wide expression profil- cer cells harboring heterozygous CBP mutations; however, ing data support these findings: expression of genes involved it remains unclear whether mutant CBP protein was also in cell growth and apoptosis-related pathways was signifi- recruited, because the WT and mutant CBP proteins could cantly altered by p300 depletion specifically inCBP- deficient not be distinguished using the available antibodies. More cancer cells. Furthermore, we showed that CBP and p300 over, SU-DHL-5 cells, which have a hemizygous focal CBP HAT activity contributes to positive transcriptional regula- deletion that leaves the remaining allele intact, were also sen- tion of the MYC gene by redundantly localizing upstream sitive to C646. Thus, not only cancer cells with CBP-deficient of the MYC gene TSS and acetylating histones H3K18 and mutations, but also those with hemizygous CBP deletion, H3K27, thereby promoting transcription initiation. Thus, might be vulnerable to p300-inhibitory therapy. Because p300 depletion–mediated transcriptional suppression of C646 is an experimental drug that is not intended for use as MYC, caused by impairment of histone acetylation at the a medication, more active p300-HAT inhibitors are required MYC TSS, contributes to the vulnerability of CBP-deficient for use in the cancer clinic. We are currently attempting to cancer cells. On the other hand, expression of a number develop such compounds. In addition, our results indicated of genes involved in cell growth and apoptosis pathways, that bromodomain inhibitors with high specificity for p300 including E2F1, E2F2, and its transcriptional targets CCND1 could also be clinically useful. and CDC45, was suppressed upon depletion of p300 in CBP- In summary, we developed a novel therapeutic strategy for deficient cancer cells. The transcriptional suppression of targeting CBP-deficient cancers and clearly demonstrated the these genes was rescued by exogenous MYC expression, and therapeutic potential of this strategy in lung and hematopoi- the impaired growth was rescued only by MYC, suggesting etic cancer cells. Our results revealed that CBP was responsi- that MYC plays key roles in the vulnerability of CBP-defi- ble for vulnerability to p300 depletion via a synthetic-lethal cient cancer cells to p300 addiction. interaction between the two genes. According to the data CBP and p300 colocalize with acetylated H3K27 in pro- in The Cancer Genome Atlas, many cancers harboring CBP moter regions, whereas BRD4 also colocalizes with acetylated mutations carry an intact p300 gene, indicating that the syn- H3K27 on the MYC gene promoter, suggesting that these thetic-lethal relationship between the CBP and p300 genes is proteins could contribute to the upregulation of MYC gene conserved in other types of cancer (Supplementary Table S4; expression (50–52). In fact, cancer cells with MYC-activat- P value for mutually exclusivity = 1.0 × 10−7; Fisher exact test). ing genomic aberrations are sensitive to BRD4 inhibitors, However, it is possible that mutations in other genes involved which cause downregulation of MYC expression and induce in chromatin regulation might also affect this vulnerability,

G1 arrest and/or apoptosis (53–55). These consequences are because cancer genomes frequently contain aberrations in a quite similar to those induced by p300 suppression in CBP- variety of those genes. In fact, a recent study newly identified deficient cancer cells. On that basis, we conclude that cancer KAT6B as a gene that is frequently deficient in lung cancer cells driven by deleterious CBP aberrations have acquired a (38). Therefore, to obtain a complete picture of vulnerability specific cellular context that is highly dependent onMYC to depletion of p300 and other HATs, further studies are war- expression for survival, as in the case of MYC-driven tumors. ranted. A subset of diffuse large B-cell lymphomas (DLBCL)

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RESEARCH ARTICLE Ogiwara et al. harbor activating mutations in the EZH2 gene, which encodes with the CD4 Enrichment Kit (Life Technologies). siRNA transfection a histone methyltransferase (56, 57), and such cells are sensi- was performed using the Lipofectamine RNAiMAX reagent (Invitro- tive to EZH2 inhibitors (57–59). However, EZH2 mutations gen). The siRNAs used in this study are listed in Supplementary Table are mutually exclusive with CBP mutations. Similarly, CBP S6. Antibodies used in this study are listed in Supplementary Table S7. mutations in lung squamous cell carcinomas and stomach C646 was purchased from Tocris Bioscience. L002, SGC-CBP30, and I-CBP112 were purchased from Sigma Aldrich. adenocarcinomas are mutually exclusive with amplification of druggable genes such as FGFR1 and ERBB2 (Supplemen- siRNA Library Screen tary Fig. S8). Thus, p300-inhibitory therapy holds promise Noncancerous cells (HFL1 and MRC5), CBP WT cancer cells as a “synthetic lethal–based therapy” for treating a variety of (A549), and CBP-deficient cancer cells (LK2) were used in screening human cancers with deleterious CBP aberrations. Notably, assay. Lack of mutations in the CBP, p300, SMARCA4, ARID1A, and however, a small subset (<1%) of tumors have mutations in ARID2 genes in the HFL1, MRC5, and A549 cell lines, and the pres- both the CBP and p300 genes, as shown in Supplementary ence of homozygous CBP deletion and absence of mutations in the Fig. S8, and such coinactivation, probably accomplished by other genes in the LK2 cell line, were verified by targeted genome functional complementation by other HATs, may represent capture and massively parallel sequencing using a MiSeq sequencer a mechanism of resistance to synthetic-lethal therapy, as dis- and a 90-gene targeted panel, the NCC oncopanel (Cat No. 931196; cussed in the context of BRM/SMARCA2 depletion therapy Agilent). Cells seeded in 96-well plates were transfected with 50 against cancers with BRG1/SMARCA4 deficiency (19, 60). This nmol/L siRNAs targeting genes related to chromatin regulation (pools of three different siRNAs targeted each gene; Ambion) using issue should also be investigated further. the Lipofectamine RNAiMAX reagent (Invitrogen). Cell viability was assessed after 5 days using the CellTiter-Glo Luminescent Cell Viabil- METHODS ity Assay (Promega). The luminescence reading for each well on Materials duplicate plates was expressed relative to the luminescence value of wells transfected with nontargeting siRNA. A result was considered The lung cancer cell lines H322, H157, H1703, H520, H1299, a potential synthetic-lethal hit if survival under the simultaneous H2009, and H2087 were provided from the establishers, Drs. John D. knockdown condition was less than 35% in CBP-deficient cells and Minna and Adi F. Gazdar (The University of Texas Southwestern Medi- more than 65% in noncancerous or CBP WT cells. cal Center, Dallas, TX) in 2001, and the KM12C colon cancer cell line was provided from the establisher, Dr. Isaiah J. Fidler (The University Cell Proliferation Assay of Texas MD Anderson Cancer Center, Houston, TX) in 2000. The Cell proliferation was examined by measuring cellular ATP levels LK2 lung and LoVo colon cancer cell lines were purchased from the using the CellTiter-Glo Luminescent Cell Viability Assay. To meas- Japanese Collection of Research BioResources (JCRB) Cell Bank in ure cell proliferation after siRNA-mediated knockdown, cell lines April 2001 and August 1989, respectively. A lung cell line, SQ5, and were transfected with siRNAs (20–50 nmol/L) using Lipofectamine two esophagus cancer cell lines, TE8 and TE10, were purchased from RNAiMAX. After 48 hours, the cells were trypsinized, counted, and RIKEN BioResource Center (RBC) Cell Bank in April 2001 and June reseeded at a specified density in 96-well plates. To measure cell pro- 2014, respectively. The lung cancer cell lines A549 (1990), H661 (May liferation after treatment with C646, cells were trypsinized, counted, 2007), and H1048 (June 2014); the colon cancer cell lines HCT116, reseeded at a specified density in 96-well plates, and then exposed HT-29, KM12C, SW480, and SW620 (2000); the lymphoma cell lines to the indicated concentrations of C646, SGC-CBP30, or I-CBP112. Jurkat (2011), Loucy, Farage, and RL (June 2013); an embryonic kidney Cell viability was measured using the CellTiter-Glo Luminescent Cell cell line, HEK293T (July 2013); fetal lung fibroblast cell lines HFL1 and Viability Assay; luminescence was measured in an Envision Multi- MRC5 (June 2011); and a hTERT-immortalized retinal pigment epi- label plate reader (PerkinElmer). thelial cell line, hTERT_RPE-1 (June 2013), were purchased from the ATCC. Lymphoma cell lines RC-K8, SU-DHL-5, SU-DHL-6, U-2932, Colony Formation Assay VAL, and WSU-NHL were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) in June 2011. We authen- The effect of siRNA-KD on cancer cell survival was evaluated in ticated the A549, H1703, H2009, H2087, H322, LK2, H157, H520, colony formation assays. Briefly, cancer cell lines were transfected H661, H1299, and SQ5 cell lines by verifying known cancer-related with siRNAs (20–50 nmol/L) using Lipofectamine RNAiMAX. After gene alterations by Sanger sequencing in 2009 (61). We authenticated 48 hours, the cells were trypsinized, counted, reseeded at a specified the HFL1, MRC5, A549, H1703, LK2, H157, H520, H661, H1299, and density in 6-well dishes, and cultured for a further 10 to 14 days to HCT116 cell lines in 2013 (19) and the KM12C, Jurkat, SU-DHL-5, allow colony formation. To measure cell survival after treatment VAL, and WSU-NHL cell lines in 2013–2014 by verifying alterations with C646, SGC-CBP30, I-CBP112, and L002, cells were trypsinized, of multiple cancer-related genes by target sequencing using the Ion counted, reseeded at a specified density in 6-well plates, and then PGM system. Other cell lines have not been further authenticated by exposed to the indicated concentrations of the drugs for 10 to 14 us. We used cell lines for assays within 3 months after resuscitation. days. The cells were then fixed for 5 minutes in 50% (v/v) methanol/ Supplementary Table S5 shows the status of CBP, p300, and other 0.01% (w/v) crystal violet. genes involved in chromatin remodeling/modification in these cells. Cell lines were cultured in RPMI-1640 (Wako) or DMEM (Wako) Genome-Wide Gene Expression Profiling supplemented with 10% FBS (GIBCO; Life Technologies), 2 μmol/l CBP and p300 WT H1299 cancer cells, CBP-KO H1299 cells, and glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Wako). p300-KO H1299 cells were transfected with siRNAs (siNT, sip300 H1299 CBP-KO cells (2G2) bearing an insertion of the puromycin D1, siCBP D2) using the Lipofectamine RNAiMAX reagent. After 48 resistance gene in exon 2 of CBP were constructed using the TALEN hours, total RNA was extracted using the Qiagen RNeasy kit. The system (Life Technologies). HEK293T CBP-KO cells (#2–4) bearing integrity of the extracted RNA was confirmed by NanoDrop spec- a 1-bp insertion in exon 9 of CBP were constructed using the Edit-R trophotometry (NanoDrop Technologies). Total RNA was reverse- CRIPSR-Cas9 Genome Engineering system (GE Healthcare Dharma- transcribed using the Agilent Low Input Quick Amp Labeling Kit con). H1299 p300-KO cells (#23) bearing a 26-bp deletion in exon 1 (Agilent Technologies). cDNA was hybridized in duplicate to Agilent of p300 were constructed using the GeneArt CRISPR Nuclease Vector microarrays (SurePrint G3 Human Gene Expression 8 × 60K Ver.1.0,

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Targeting p300 Addiction in CBP-Deficient Cancers RESEARCH ARTICLE

G4851: 42,405 probes) using a Gene Expression Hybridization Kit (HAT-) 23254; MYC: pcDNA3-cmyc (Addgene); MAX: pLVX-MAX; (Agilent Technologies) for 16 hours at 65°C. After the arrays were kindly gifted from Dr. Montse Sanchez-Cespedes; ref. 62), or by washed using the Gene Expression Wash Pack (Agilent Technolo- establishing cells stably harboring plasmids expressing cDNAs (MYC: gies), the data were extracted using an Agilent scanner. The arrays pLOC-MYC; CCND1: pLOC-CCND1; CDC45: pLOC-CCND1; were first analyzed using the Feature Extraction software (Agilent Thermo Scientific). In one transient transfection method, cells were Technologies). A quantitative signal and qualitative detection call seeded in 12-well plates and transfected the next day with 1 μg of were generated for each sample and transcript. plasmid DNA using Lipofectamine 3000 (Invitrogen). Twelve hours Data files were subsequently analyzed utilizing GeneSpring GX12.6 after transfection, the cells were further transfected with siRNAs (25 (Agilent Technologies). The raw expression data of 42,545 probe sets nmol/L) using Lipofectamine RNAiMAX. In other transient transfec- on SurePrint G3 Human Gene Expression arrays were processed and tions, cells were seeded in 12-well plates and transfected with both log2-transformed. Probe sets with average log2 expression levels less plasmid DNA (1 μg) and siRNAs (25 nmol/L) using Dharmafect Duo than 4 were considered quantifiable, and therefore were subjected to (GE Healthcare Dharmacon). In stably expressing cells, cells were the analysis for the remaining 42,485 probe sets. Expression data for seeded in 12-well plates and subsequently transfected with siRNAs each sample were normalized to their median expression values in (25 nmol/L) using Lipofectamine RNAiMAX. After 48 hours, the cells the siNT-treated condition. Genes were grouped by fold changes as were subjected to proliferation or colony formation assays. described in Fig. 2A. All raw microarray data files have been deposited in the Gene Expression Omnibus (GSE73682). Cell-Cycle Analysis Cells were trypsinized, centrifuged, and washed in PBS, and then Quantitation of mRNA and Protein fixed in ice-cold 70% ethanol. The cells were then centrifuged again Cells were transfected with siRNAs and incubated for 48 hours. and incubated with PBS containing 200 μg/mL RNase A and 5 μg/mL mRNA was extracted, and cDNA was synthesized using the Super- propidium iodide (PI). Cell-cycle analysis was performed on a Guava Prep Cell Lysis and RT Kit for qPCR (Toyobo). Aliquots of cDNA were flow cytometer (Millipore). subjected to quantitative PCR using the SuperPrep/THUNDERBIRD Probe qPCR Set (Toyobo) and TaqMan Gene Expression Assays (Life Annexin V/PI Staining Assay for Apoptosis Technologies); the gene-specific primer/probe sets are listed in Sup- The Annexin V–FITC/PI apoptosis detection kit (Roche) was used plementary Table S8. PCR was performed in an ABI ViiA7 Real-Time to detect apoptotic cells. Briefly, the cell pellet was suspended in ×1 PCR System (Life Technologies) under the following conditions: binding buffer, followed by incubation with Annexin V–FITC and PI denaturation at 95°C for 15 seconds, followed by annealing and in the dark for 10 minutes. Fluorescence was then analyzed by flow extension at 60°C for 60 seconds (45 cycles). For each sample, the cytometry. mRNA levels of the target genes were normalized against the level of GAPDH. The target gene/GAPDH ratios were then normalized ChIP Assay −ΔΔCt against the corresponding ratio from siNT cells using the 2 ChIP assays were performed as previously described (1). Briefly, method. All experiments were performed in triplicate, and data are 1 × 106 cells were harvested 48 hours after transfection with siRNA expressed as means ± SD. Intensities of acetylated-H3 protein signals or treatment with C646, and then treated with 1% formaldehyde were calculated using the Multi Gauge software V3.1 (Fujifilm). for 10 minutes at room temperature to cross-link proteins to DNA. Glycine (0.125 mol/L) was added to stop the cross-linking process. Generation of shRNA and cDNA Expression Lentiviruses ChIP assays were performed using the ChIP-IT Express Enzymatic and Virus-Infected Cells Kit (Active Motif) and normal IgG (Cell Signaling Technology), or The shRNA-expressing lentiviral vector pTRIPZ (shNT, OHS5832; antibodies against CBP (Santa Cruz Biotechnology), p300 (Santa shp300, RHS4696-101353787; Open Biosystems) and the Trans- Cruz Biotechnology), histone H3K18 (Active Motif), histone H3K27 Lentiviral Packaging System (Open Biosystems) were used for tet- (Active Motif), or RNA polymerase II (Active Motif). Purified DNA inducible shRNA expression. shRNA-expressing lentiviral vectors was subjected to quantitative PCR using primer pairs listed in Sup- (shLuc, pLKO.1-shLuciferase; shp300-1/2, pLKO.1-shp300-1/2; a plementary Table S9 and the SuperPrep/THUNDERBIRD SYBR kind gift from Dr. Chia-Hsin Chan; Open Biosystems) and the qPCR Set (Toyobo). PCR conditions were as follows: denaturation at ViraPower Lentiviral Expression System (Life Technologies) were 95°C for 15 seconds, followed by annealing and extension at 60°C used to achieve stable shRNA expression. The cDNA-expressing for 60 seconds (45 cycles). PCR was performed on an ABI 7900HT lentiviral vector pLOC (MYC: pLOC-MYC; CCND1: pLOC-CCND1; Fast Real-Time PCR System (Life Technologies). Protein enrichment CDC45: pLOC-CCND1; Thermo Scientific) and packaging plasmids was expressed as a percentage of input. All experiments were per- (psPAX2: #12260 and pMD2.G: #12259; Addgene) were used for con- formed in triplicate, and data are expressed as means ± SD. stitutive expression of cDNAs. To generate virus, HEK293T, 293LTV or 293FT cells were transfected with lentiviral plasmids and packag- Mouse Xenograft Model ing plasmids using Lipofectamine 3000 (Invitrogen); on the follow- Cells were counted and resuspended in a 1:1 mixture of culture ing day, the medium was replaced with fresh growth medium, and medium and Matrigel (BD Biosciences) on ice. The cells were then lentivirus-containing supernatants were harvested and concentrated injected (0.5–2 × 106 cells/mouse for H1299 and LK2; 0.5–1 × 107 by centrifugation. To establish cells infected with viral constructs, cells/mouse for Jurkat cells) subcutaneously into the flank or thoracic cells were transduced with lentiviral vectors, and then incubated for 7 cavity of 7-week-old female BALB/c-nu/nu mice (CLEA, Japan) using to 14 days in growth medium containing 2 μg/mL puromycin (Sigma a protocol approved by the Ethical Committee on Animal Experi- Aldrich) or 20 μg/mL blasticidin (Wako). To induce the expression of ments at the National Cancer Center. In the subcutaneous model, shRNA in tet-inducible cell lines, cells were treated with doxycycline once the tumors were palpable (about 14–20 days after implantation), (0.5–1 μg/mL; Selleck) for 72 to 96 hours. the mice were randomly divided into two groups. For the doxycycline treatment method, mice were fed either a diet containing doxy- Complementation Assay cycline (400 ppm) or a control diet. In the drug treatment method, Rescue of the proliferation of CBP-KO cells by complementa- mice were injected intraperitoneally with C646 (25 mg/kg; Tocris) tion was performed by transiently transfecting plasmids expressing once a day for 14 days. Tumor growth was measured every 3 to 4 days cDNAs (p300: pcDNA3.1-p300 23252; p300 HAT: pcDNA3.1-300 using calipers. In the orthotopic model (63), LK2 shp300 cells were

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RESEARCH ARTICLE Ogiwara et al. treated with or without doxycycline for 4 days, and then subsequently 3. Wu YM, Su F, Kalyana-Sundaram S, Khazanov N, Ateeq B, Cao X, injected into the thoracic cavity followed by feeding diet containing et al. Identification of targetable FGFR gene fusions in diverse can- doxycycline (400 ppm) or control diet. Tumor growth was measured cers. Cancer Discov 2013;3:636–47. 24 days after injection. The volume of the implanted tumors was 4. Singh D, Chan JM, Zoppoli P, Niola F, Sullivan R, Castano A, et al. calculated using the formula V = L × W2/2, where V is volume (mm3), Transforming fusions of FGFR and TACC genes in human glioblas- L is the largest diameter (mm), and W is the smallest diameter (mm). toma. Science 2012;337:1231–5. At the end of the experiment, the mice were sacrificed in accordance 5. Weiss J, Sos ML, Seidel D, Peifer M, Zander T, Heuckmann JM, et al. with standard protocols. Frequent and focal FGFR1 amplification associates with therapeutically tractable FGFR1 dependency in squamous cell lung cancer. Sci Statistical Analysis Transl Med 2010;2:62ra93. 6. Peifer M, Fernandez-Cuesta L, Sos ML, George J, Seidel D, Kasper LH, All experiments were performed in triplicate, and data are expressed et al. Integrative genome analyses identify key somatic driver muta- as means ± SD. Data from mouse xenograft models are expressed as tions of small-cell lung cancer. Nat Genet 2012;44:1104–10. means ± SE. Differences between drug-treated and untreated cells 7. Banerji S, Cibulskis K, Rangel-Escareno C, Brown KK, Carter SL, Fre- were evaluated using the Student t test. Statistically significant differ- derick AM, et al. Sequence analysis of mutations and translocations ences are indicated by asterisks (*, P < 0.05; **, P < 0.01). across breast cancer subtypes. Nature 2012;486:405–9. 8. Carpten JD, Faber AL, Horn C, Donoho GP, Briggs SL, Robbins CM, Disclosure of Potential Conflicts of Interest et al. A transforming mutation in the pleckstrin homology domain of No potential conflicts of interest were disclosed. AKT1 in cancer. Nature 2007;448:439–44. 9. Takeuchi K, Soda M, Togashi Y, Suzuki R, Sakata S, Hatano S, et al. Authors’ Contributions RET, ROS1 and ALK fusions in lung cancer. Nat Med 2012;18:378–81. 10. Lipson D, Capelletti M, Yelensky R, Otto G, Parker A, Jarosz M, et al. Conception and design: H. Ogiwara Identification of new ALK and RET gene fusions from colorectal and Development of methodology: H. Ogiwara lung cancer biopsies. Nat Med 2012;18:382–4. Acquisition of data (provided animals, acquired and managed 11. Kohno T, Ichikawa H, Totoki Y, Yasuda K, Hiramoto M, Nammo patients, provided facilities, etc.): H. Ogiwara, M. Sasaki, T. Oike, T, et al. KIF5B-RET fusions in lung adenocarcinoma. Nat Med S. Higuchi, Y. Tominaga 2012;18:375–7. Analysis and interpretation of data (e.g., statistical analysis, 12. Rikova K, Guo A, Zeng Q, Possemato A, Yu J, Haack H, et al. Global biostatistics, computational analysis): H. Ogiwara survey of phosphotyrosine signaling identifies oncogenic kinases in Writing, review, and/or revision of the manuscript: H. Ogiwara, lung cancer. Cell 2007;131:1190–203. T. Kohno 13. Vaishnavi A, Capelletti M, Le AT, Kako S, Butaney M, Ercan D, et al. Administrative, technical, or material support (i.e., reporting or Oncogenic and drug-sensitive NTRK1 rearrangements in lung cancer. organizing data, constructing databases): H. Ogiwara, T. Mitachi Nat Med 2013;19:1469–72. Study supervision: H. Ogiwara, T. Kohno 14. Kandoth C, McLellan MD, Vandin F, Ye K, Niu B, Lu C, et al. Mutational landscape and significance across 12 major cancer types. Acknowledgments Nature 2013;502:333–9. 15. Alexandrov LB, Nik-Zainal S, Wedge DC, Aparicio SA, Behjati S, The authors thank Kazuaki Takahashi, Hirokazu Ishikawa, Biankin AV, et al. Signatures of mutational processes in human can- Hitoshi Ichikawa, Takashi Kubo, Ayaka Otsuka, Yuko Fujiwara, and cer. Nature 2013;500:415–21. Naoto Tsuchiya for technical assistance and helpful comments, and 16. Kadoch C, Hargreaves DC, Hodges C, Elias L, Ho L, Ranish J, Chia-Hsin Chan and Montse Sanchez-Cespedes for providing plas- et al. Proteomic and bioinformatic analysis of mammalian SWI/SNF mids. They thank Scientific Support Programs for Cancer Research complexes identifies extensive roles in human malignancy. Nat Genet Grant-in-Aid for Scientific Research on Innovative Areas Ministry of 2013;45:592–601. Education, Culture, Sports, Science, and Technology for the genotyp- 17. Medina PP, Sanchez-Cespedes M. Involvement of the chromatin- ing analysis of cell lines. remodeling factor BRG1/SMARCA4 in human cancer. Epigenetics 2008;3:64–8. Grant Support 18. Oike T, Ogiwara H, Nakano T, Kohno T. Proposal for a synthetic This study was supported in part by Grants-in-Aid for Scientific lethality therapy using the paralog dependence of cancer cells– Research on Innovative Areas (22131006) from the Ministry of Edu- response. Cancer Res 2014;74:4948–9. 19. Oike T, Ogiwara H, Tominaga Y, Ito K, Ando O, Tsuta K, et al. A cation, Culture, Sports, Science, and Technology of Japan; Grants- synthetic lethality-based strategy to treat cancers harboring a genetic in-Aid for Young Scientists (B) KAKENHI (23701110 and 26830122) deficiency in the chromatin remodeling factor BRG1. Cancer Res from the Japan Society for the Promotion of Science; and a collabora- 2013;73:5508–18. tion research grant from Daiichi-Sankyo Co., Ltd. 20. D’Antonio M, Guerra RF, Cereda M, Marchesi S, Montani F, Nicassio The costs of publication of this article were defrayed in part by F, et al. 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Targeting p300 Addiction in CBP-Deficient Cancers Causes Synthetic Lethality by Apoptotic Cell Death due to Abrogation of MYC Expression

Hideaki Ogiwara, Mariko Sasaki, Takafumi Mitachi, et al.

Cancer Discov Published OnlineFirst November 24, 2015.

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