Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2020 Mar 15.
Published in final edited form as: Cancer Res. 2019 Jan 23;79(6):1191–1203. doi: 10.1158/0008-5472.CAN-18-3223

BET inhibitors potentiate chemotherapy and killing of SPOP-mutant colon cancer cells via induction of DR5

Xiao Tan 1,2,3, Jingshan Tong 2,3, Yi-Jun Wang 2,3, Rochelle Fletcher 2,3, Robert E Schoen 2,4, Jian Yu 2,5, Liangfang Shen 1,*, Lin Zhang 2,3,*
PMCID: PMC6420862  NIHMSID: NIHMS1519475  PMID: 30674532

Abstract

Bromodomain and extra-terminal domain (BET) family proteins such as BRD4 are epigenetic readers that control expression of a number of oncogenic proteins. Targeting this family of proteins has recently emerged as a promising anticancer approach. BET inhibitors (BETi), either alone or in combination with other anticancer agents, have exhibited efficacy in a variety of tumors. However, the molecular mechanisms underlying differential response to BETi are not well understood. In this study, we report that Death Receptor 5 (DR5), a key component of the extrinsic apoptotic pathway, is markedly induced in response to BRD4 depletion and BETi treatment in colorectal cancer (CRC) cells. Induction of DR5 following BET inhibition was mediated by endoplasmic reticulum (ER) stress and CHOP-dependent transcriptional activation. Enhanced DR5 induction was necessary for the chemosensitization and apoptotic effects of BETi and was responsible for increased BETi sensitivity in CRC cells containing a mutation in Speckle-type POZ protein (SPOP), a subunit of BRD4 E3 ubiquitin ligase. In a CRC xenograft model, BETi combined with chemotherapy suppressed tumor growth in a DR5-dependent manner and potently inhibited patient-derived xenograft (PDX) tumor growth with enhanced DR5 induction and apoptosis. These findings suggest that BETi alone or in combination with chemotherapy is effective against CRC due to enhanced DR5 induction and apoptosis. DR5 induction may also serve as a useful marker for designing personalized treatment and improved CRC combination therapies.

Significance

Findings reveal how BET inhibition sensitizes chemotherapy and kills a subset of colon cancer cells with specific genetic alterations and may provide a new molecular marker for improving colon cancer therapies.

Keywords: BET inhibitors, DR5, SPOP, colorectal cancer, apoptosis

Introduction

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the US (1). CRC patients are typically treated with conventional chemotherapeutic drugs including 5-fluorouracil (5-FU), oxaliplatin, and irinotecan (2), which have limited specificity and efficacy in advanced CRCs (3,4). CRC patients are also treated with targeted drugs such as the anti-EGFR antibodies cetuximab and panitumumab, and the multi-kinase inhibitor regorafenib (2). Nonetheless, tumors treated with targeted drugs almost invariably develop resistance shortly after initial therapy (5). The benefit of anti-PD-1 immunotherapy is limited in 10–15% of CRCs deficient in DNA mismatch repair (MMR) (6). There is a critical need for developing novel therapies against CRCs.

Epigenetic alterations such as DNA hypermethylation and histone modifications play an important role in CRC progression (7,8). The bromodomain and extra-terminal domain (BET) family proteins, including BRD2, BRD3, BRD4 and BRDT, are epigenetic readers of acetylated histone marks (9,10). They function as transcriptional co-activators by recognizing acetylated lysine residues through their bromodomains to regulate transcriptional elongation by RNA polymerase II (10). BET family proteins are frequently assembled into super enhancers and control the expression of oncogenes such as c-Myc (11). Recent studies show that BRD4, the most extensively characterized BET protein, is critical for CRC cell proliferation (12,13).

Targeting the BET family proteins has recently emerged as a promising anticancer approach (14). BET inhibitors (BETi), alone or in combination with other anticancer agents, have exhibited efficacy in a variety of tumors (14). Recent studies revealed that mutations in Speckle-type POZ protein (SPOP), encoding a component of the E3 ubiquitin ligase of BET proteins, dictate BETi sensitivity in some cancer cells (15–17). While SPOP-inactivating mutations in prostate cancer cells stabilize BET proteins and confer resistance to BETi, SPOP-activating mutations sensitize endometrial cancer cells to BETi (15–17). The molecular basis of resistance and differential BETi sensitivity caused by SPOP mutations is unclear.

Cell killing is a key mechanism of anticancer therapies (18). BETi sensitivity was shown to be mediated by the induction of apoptosis (19,20), which is regulated by the extrinsic (death receptor) and intrinsic (mitochondrial) pathways. The extrinsic pathway is engaged upon activation of the TNF family receptors such as death receptor 5 (DR5; TRAILR2; TNFRSF10B) and DR4, which further recruit other proteins to activate caspase 8 and downstream caspases (21). DR5 can also be induced by p53 upon DNA damage (22), or by C/EBP homologous protein (CHOP) in response to endoplasmic reticulum (ER) stress (23). The mitochondrial pathway is activated by the Bcl-2 family members via mitochondrial dysfunction (24,25). Relative to the mitochondrial pathway, the role of the extrinsic pathway in anticancer therapies is less well characterized.

In this study, we investigated the molecular basis of differential response to BETi in CRC cells. Our results suggest that DR5-mediated apoptosis plays a critical role in chemosensitization by BETi in CRC cells, and is responsible for increased BETi sensitivity in CRC cells with SPOP-activating mutations.

Materials and Methods

Cell culture and treatments

Human CRC cell lines, including HCT116, HT29, RKO, SW48, Lim1215, and NCI-H508, were purchased from the American Type Culture Collection and cultured in McCoy’s 5A modified media (Invitrogen). SNU-407 was purchased from AddexBio and cultured in RPMI 1640 (Invitrogen). DR5-knockout (KO) HCT116 cells were previously described (26). DR5-KO RKO cells were generated by CRISPR/Cas9 using a single guide RNA sequence 5’-CGCGGCGACAACGAGCACAA-3’ as described (27). Cells were authenticated in 2018 by genotyping and analysis of protein expression by western blotting, and routinely checked for Mycoplasma contamination by PCR. All cell lines were maintained at 37°C and 5% CO2 atmosphere. Cell culture media were supplemented with 10% defined FBS (HyClone), 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). For drug treatment, cells were plated in 12-well plates at 20–30% density 24 hr before treatment. DMSO (Sigma) stocks of JQ1, I-BET151 (Apexbio), OTX015, I-BET762, 5-FU, and oxaliplatin (Sigma) were prepared and diluted in cell culture media before adding to cells.

Transfection and small interfering RNA (siRNA) knockdown

Cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. pDONR223 vectors expressing wild-type (WT; plasmid #81856), E47A (plasmid #81645), and E50K (plasmid #81631) mutant SPOP were previously described (28), and obtained from Addgene. SiRNA transfection was performed 24 hr before drug treatment using 200 pmole of control scrambled, CHOP (5’-GCACAGCUAGCUGAAGAGAdTdT-3’), DR5 (5’-AAGACCCUUGUGCUCGUUGUCdTdT-3’) (Dharmacon), BRD4 (sc-43639), or SPOP (sc-63056) siRNA (Santa Cruz Biotechnology).

mRNA sequencing (RNA-Seq)

Total RNA was prepared from HCT116 cells transfected with either control scrambled or BRD4 siRNA for 24 hr using the Quick-RNA Kit (Zymo Research) according to manufacturer’s instructions. Library construction, RNA sequencing, and data analysis were performed by Novogene using the Illumina HiSeq platform. Sample quality was assessed by HTSeq v0.6.1 to count the read numbers mapped of each gene. Data quality was ensured by the percentage of bases with a sequencing quality score above Q30. FPKM (fragments per kilobase of transcript per million mapped reads) of each gene was calculated based on the length of a gene and read counts mapped to this gene. Differential expression analysis was performed using the DESeq R package (2_1.6.3).

Western blotting

Western blotting was performed as previously described (29) using antibodies listed in Table S1.

Real-time reverse transcriptase (RT) PCR

Total RNA was isolated from cells using the Mini RNA Isolation II Kit (Zymo Research) according to the manufacturer’s protocol. One μg of total RNA was used to generate cDNA using the SuperScript II reverse transcriptase (Invitrogen). PCR was performed with previously described cycle conditions (30) and primers (23), except for CHOP (5’-GGTCCTGTCTTCAGATGAAAATG-3’/5’-CAGCCAAGCCAGAGAA GCA-3’).

MTS assay

Cells seeded in 96-well plates at a density of 1×104 cells/well were treated with different agents for 72 hr. Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (Promega) according to the manufacturer’s instructions. Chemiluminescence was measured using a Wallac Victor 1420 Multilabel Counter (Perkin Elmer). Each assay was conducted in triplicate and repeated three times.

Luciferase assay

pGL3-based DR5 luciferase reporter constructs containing WT or mutant CHOP-binding site were previously described (31,32). To measure reporter activities, cells were transfected with WT or mutant DR5 reporter along with the transfection control β-galactosidase reporter pCMVβ (Promega). Cell lysates were collected and luciferase activities were measured and normalized as previously described (33). All reporter experiments were performed in triplicate and repeated three times.

Chromatin immunoprecipitation (ChIP)

ChIP was performed using the Chromatin Immunoprecipitation Assay Kit (EMD Millipore) according to the manufacturer’s instructions. The precipitates were analyzed by PCR for DR5 promoter using the primer pair 5’-AGGTTAGTTCCGGTCCCTTC-3’/5’-CAACTGCAAATTCCACCACA-3’.

Apoptosis assays

Apoptosis was measured by counting cells with condensed and fragmented nuclei after nuclear staining with Hoechst 33258 (Invitrogen) as previously described (33). At least 300 cells were analyzed for each group. Apoptosis was also analyzed by flow cytometry of cells stained with Annexin V/propidium iodide (PI) (33). Viable cells in 12-well plates after drug treatment for 36 hr were visualized by staining with crystal violet (Sigma). Colony formation was assayed by plating equal numbers of treated cells in 12-well plates at appropriate dilutions, followed by visualization of colonies by crystal violet staining after 14 days, and counting of colony numbers.

Cell line and patient-derived xenograft (PDX) tumor experiments

All animal experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Female 5–6-week old Nu/Nu mice (Charles River) and 4–6-week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice (Jackson Laboratory) were housed in a sterile environment with micro isolator cages and allowed access to water and chow ad libitum.

Cell line xenografts were established by subcutaneously injecting 4×106 WT or DR5-KO HCT116 cells into both flanks of Nu/Nu mice. Tumors were allowed to grow for 7 days before treatment. Tumor-bearing mice were randomized into 4 groups and subjected to the following treatments for 3 weeks: 1) untreated; 2) JQ1 (i.p.; 30 mg/kg daily); 3) 5-FU (i.p.; 25 mg/kg every other day); and 4) JQ1+5-FU. JQ1 (AstaTech) was dissolved in DMSO to 50 mg/ml and then diluted 1:10 in 10% hydroxypropyl-β-cyclodextrin (Sigma). Injectable 5-FU solution (APP Pharmaceuticals) was diluted with Hank’s Balanced Salt Solution (Invitrogen). Six mice were used for each group.

Two PDX models were established using treatment-naïve primary tumors resected from patients with newly diagnosed CRCs under written informed consent and approved IRB protocol. PDX1 was established from a KRAS-mutant (G13D), NRAS-mutant (G12D), and MMR-proficient tumor (T4N0M1) in the sigmoid colon of a 77-year-old male. PDX2 was established from an MMR-proficient tumor (T2N0) in the right colon of a 69-year-old female. Tumors were implanted subcutaneously and passaged as described (34). Briefly, de-identified patient CRC samples were delivered to the laboratory in 1× Antibiotic/Antimycotic solution (Invitrogen) within 4 hr after tumor resection. Tissues were cut into 25-mg pieces and directly implanted subcutaneously into both flanks of NSG mice. Tumors passaged and expanded for two generations (P2) in NSG mice were used for the described experiments. Treatment was performed for 20 days in the same way as described for the cell line xenografts, with each group including 4 mice.

Tumor growth was monitored by calipers, and tumor volumes were calculated according to the formula ½ × length × width2. Ethical endpoint was defined as a time point when a tumor reached 1.5 cm or more in any dimension. Tumors were dissected and fixed in 10% formalin and embedded in paraffin. Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling (TUNEL; EMD Millipore), active caspase 3 (Cell Signaling Technology), active caspase 8 (Cell Signaling Technology), and Ki67 (Dako) immunostaining was performed on 5-μM paraffin-embedded tumor sections as previously described (33), by using an AlexaFluor 488-conjugated secondary antibody (Invitrogen) for detection and 4’ 6-Diamidino-2-phenylindole (DAPI) for nuclear counter staining.

Statistical analysis

Statistical analysis was carried out using the GraphPad Prism VI software. For cell culture experiments, P values were calculated using the Student’s t-tests. Means ± standard deviation (SD) are reported in the figures. For animal experiments, P values were calculated by repeated measures (RM) ANOVA with Fisher’s LSD post-hoc tests. Means ± standard error of the mean (SEM) are reported in the figures. Differences were considered significant if P < 0.05.

Results

BETi induce DR5 expression through CHOP-mediated transcriptional activation.

To determine the effects of BET inhibition in CRC cells, RNA-Seq was performed on HCT116 cells transfected with BRD4 or control scrambled siRNA. Analysis of differentially expressed genes revealed a marked induction of DR5 mRNA (FKPM 34.78 vs. 15.63; P < 0.01) and two isoforms of DR5 protein upon BRD4 knockdown (Fig. 1A). Treating HCT116 cells with different BETi, including JQ1, OTX015, I-BET151, and I-BET762, induced DR5 in a dose- and time-dependent manner (Fig. 1, B and C). DR5 induction by BRD4 knockdown and BETi treatment was observed in other CRC cell lines with different CRC driver mutations (Table S2), including those of KRAS, BRAF, PIK3CA, p53, SMAD4, SPOP and FBW7, and associated with reduced c-Myc expression (Fig. 1, D and E). The fold changes of both isoforms of DR5 protein were found to be higher than those of DR5 mRNA (Fig. 1A, 1C and 1E), suggesting a more prominent role of DR5 protein induction in determining response to BETi. Furthermore, other members of the death receptor family, including DCR1, DCR2, TNFR1 and TNFR2, were upregulated after BRD4 knockdown or BETi treatment (Fig. 1A and S1A). In contrast, the mRNA and protein expression of DR4 did not change significantly (Fig. 1A; Fig. S1, A-C). The Bcl-2 family members including PUMA, Bim, Bad, Bid, and Bax were either downregulated or unchanged, except for Noxa (Fig. 1A). These results indicate that BETi treatment activates DR5 and the death receptor pathway in CRC cells irrespective of common drivers, and prompted us to further investigate the functional role of DR5 and the death receptor pathway.

Figure 1. BETi treatment upregulates DR5 expression in CRC cells.

Figure 1.

Open in a new tab

(A) HCT116 cells transfected with control scrambled or BRD4 siRNA were analyzed by RNA-Seq at 24 hr after transfection. Left, verification of BRD4 knockdown and DR5 induction by western blotting; right, heat map for comparing the expression of genes related to the death receptor and mitochondrial apoptotic pathways with an indication of the ratio of DR5 FPKM (fragments per kilobase of transcript per million mapped reads). (B) Western blotting of DR5 in HCT116 cells treated with the BETi JQ1, I-BET151, OTX015 or I-BET762 at indicated concentrations for 36 hr. (C) Western blot (upper) and RT-PCR (lower) analyses of DR5 expression at indicated time points in HCT116 cells treated with indicated BETi (1 μM). (D) Western blotting of indicated proteins in CRC cell lines with indicated p53, KRAS, BRAF and PIK3CA status at 24 hr after transfection with control scrambled or BRD4 siRNA. (E) Western blotting of indicated proteins (upper) and RT-PCR of DR5 (lower) in indicated CRC cell lines treated with JQ1 or OTX015 (1 μM) for 36 hr. In (A)-(E), relative expression of BRD4 and DR5 was quantified using the Image J program, normalized to that of β-actin, and expressed as a ratio relative to that in untreated cells or those transfected with control siRNA. In (C) and (E), results were expressed as means ± SD of three independent experiments. *, P <0.05; **, P <0.01.

DR5 can be induced by p53 and other transcription factors in response to various stresses (22,23). As expected, the induction of DR5 by BETi is p53-independent and unaffected in p53-deficient and -KO cells (Fig. 1D, 1E and S1D). RNA-Seq data revealed upregulation of several ER stress response genes including CHOP following BRD4 depletion (Fig. S1E), which was confirmed by protein expression (Fig. 2A). Treating CRC cells with different BETi also induced CHOP mRNA and protein (Fig. 2B and S1F), as well as other ER stress markers including BiP expression and eIF2α (Ser51) and PERK (Thr980) phosphorylation (35) (Fig. 2B). Knockdown of CHOP by siRNA abrogated the induction of DR5 mRNA and protein by JQ1 (Fig. 2C). Chromatin immunoprecipitation (ChIP) analysis showed enhanced binding of CHOP to the DR5 promoter upon JQ1 treatment (Fig. 2D). Furthermore, JQ1 exposure activated a DR5 promoter reporter containing a CHOP-binding site (31), but did not affect a similar reporter with mutations that alter the CHOP-binding site (32) (Fig. 2E). Together, these results indicate that BETi treatment induces DR5 through ER stress/CHOP-mediated transcription.

Figure 2. BETi induce DR5 expression through ER stress and CHOP-mediated transcription.

Figure 2.

Open in a new tab

(A) Western blot (left) and RT-PCR (right) analyses of DR5 and CHOP in HCT116 cells at 24 hr after transfection with control scrambled or BRD4 siRNA. (B) Western blotting of CHOP and indicated ER stress markers (upper) and RT-PCR (lower) of CHOP in indicated CRC cell lines treated with indicated BETi (1 μM) for 36 hr. p-eIF2α: phospho-eIF2α, Ser51; p-PERK: phospho-PERK, Thr980. (C) Western blotting of CHOP and DR5 (upper) and RT-PCR (lower) of DR5 in HCT116 and RKO cells transfected with control scrambled or CHOP siRNA and treated with JQ1 (1 μM) for 36 hr. (D) Chromatin immunoprecipitation (ChIP) analysis of the binding of CHOP to the DR5 promoter in HCT116 cells treated with JQ1 (1 μM) for 36 hr with IgG as a negative control. PCR was performed using primers surrounding the CHOP-binding site in the DR5 promoter, followed by analysis of PCR products by agarose gel electrophoresis. (E) HCT116 cells transfected with a luciferase reporter of the DR5 promoter containing either wild-type (WT) or mutant CHOP-binding site, along with the transfection control β-galactosidase reporter pCMVβ. After 24 hr, cells were treated with JQ1 (1 μM) for 36 hr. Upper, a schematic representation of the CHOP-binding site in the DR5 promoter with “xxxx” indicating mutated nucleotides in the mutant reporter; lower, luciferase activities of the WT and mutant DR5 promoter reporters normalized to that of pCMVβ. In (A) and (C), relative expression of BRD4 and CHOP was quantified using Image J, normalized to that of β-actin, and expressed as a ratio relative to that in cells transfected with control siRNA. In (A), (B), (C) and (E), results were expressed as means ± SD of three independent experiments. *, P <0.05; **, P <0.01.

Chemosensitization by BETi requires DR5-mediated apoptosis in CRC cells.

BRD4 knockdown or inhibition, while ineffective alone (Fig. S2, A and B), markedly suppressed CRC cell growth when combined with 5-FU or oxaliplatin (Fig. 3, A and B; Fig. S2, B and C). The combinations induced marked apoptosis in HCT116 cells, as shown by reduced cell viability, increased nuclear fragmentation, caspases 3 and 8 activation, and annexin V staining, which could be blocked by the pan-caspase inhibitor z-VAD-fmk (Fig. S2, C-G). Enhanced apoptosis by the 5-FU/BETi combination was also observed in RKO cells (Fig. S2H). Using isogenic DR5-KO HCT116 cells generated by homologous recombination (26), we found that DR5-KO markedly suppressed all signs of apoptosis, including cell viability loss, nuclear fragmentation, and caspases 3 and 8 cleavage, in response to BRD4 knockdown or JQ1 combined with 5-FU or oxaliplatin (Fig. 3, C-E; Fig. S3, A-C), and significantly improved long-term clonogenic survival (Fig. 3F). Furthermore, knockout of DR5 by CRISPR/Cas9 in RKO cells also abolished apoptosis induced by BRD4 knockdown or JQ1 in combination with 5-FU or oxaliplatin (Fig. S3, D and E). Together, these results demonstrate that the chemosensitization effects of BETi are dependent on DR5-mediated apoptosis in CRC cells.

Figure 3. Chemosensitization by BETi is dependent on DR5-mediated apoptosis.

Figure 3.

Open in a new tab

(A) MTS analysis of HCT116 cells transfected with control scrambled or BRD4 siRNA and treated with 5-FU or oxaliplatin (OXA) at indicated concentrations for 72 hr. (B) MTS analysis of HCT116 cells treated with 5-FU or OXA at indicated concentrations with or without JQ1 (1 μM) or OTX015 (1 μM) for 72 hr. (C) MTS analysis of WT and DR5-KO HCT116 cells transfected as in (A) and treated with 5-FU and OXA as in (A) and (B). (D) WT and DR5-KO HCT116 cells treated with 5-FU (15 μg/mL) or OXA (15 μM) for 36 hr with or without BRD4 knockdown as in (A) or BETi treatment as in (B). Apoptosis was analyzed by counting cells containing condensed and fragmented nuclei after nuclear staining with Hoechst 33258. (E) Western blotting of cleaved (C) caspases 3, 8 and 9 in cells treated as in (D). Relative BRD4 expression was quantified using the Image J program, normalized to that of β-actin, and expressed as a ratio relative to that in cells transfected with control siRNA. (F) Colony formation of cells treated as in (D) as analyzed by crystal violet staining. Upper, representative images of colonies; lower, enumeration of colony numbers. In (D) and (F), results were expressed as means ± SD of three independent experiments. *, P <0.05; **, P <0.01; ***, P <0.001.

SPOP-mutant CRC cells are exquisitely sensitive to BETi due to enhanced DR5 induction.

Recent studies showed that cancer cells harboring activating mutations in SPOP, a substrate-binding subunit of E3 ubiquitin ligase of BRD4, are exquisitely sensitive to BETi due to reduced BRD4 expression (16). Upon analysis of the Catalogue Of Somatic Mutations In Cancer (COSMIC) database (Table S2), we identified another CRC line, SNU-407, which contains a SPOP mutation (G75E), in addition to NCI-H508 (SPOP-E47K) (16). SNU-407 and NCI-H508 cells were more sensitive to BETi than SPOP-WT CRC cells lines, with IC50 values of JQ1 and OXT015 ranging from 0.69 to 1.05 μM, compared to 4.26 to 12.02 μM in HCT116, HT29, RKO, SW48 and Lim1205 cells (Fig. 4A). JQ1 at 0.5 μM potently suppressed colony formation of SNU-407 and NCI-H508 cells but barely affected that of HCT116 cells (Fig. 4B). Compared to SPOP-WT CRC cells, SNU-407 and NCI-H508 cells expressed lower levels of BRD4 (Fig. 4C). Interestingly, we detected strong induction of both DR5 isoforms in SNU-407 and NCI-H508 cells by JQ1 at only 0.5 μM, in contrast to an obvious DR5 induction with JQ1 at 4 μM in the SPOP-WT cells (Fig. 4D). Consistent with the strong DR5 induction, robust caspases 3 and 8 activation was found in JQ1-treated SNU-407 and NCI-H508 cells (Fig. 4E). Knockdown of SPOP in SNU-407 and NCI-H508 cells resulted in BRD4 accumulation, abrogated JQ1-induced DR5 expression, caspases 3 and 8 activation (Fig. 4E), nuclear fragmentation (Fig. 4F), and reduced JQ1 sensitivity (Fig. 4G), suggesting that increased BETi sensitivity in these cells is attributed to SPOP mutations and enhanced apoptosis induction. Consistent with the on-target CHOP and DR5 induction in SPOP-WT cells, BRD4 knockdown also induced DR5 and CHOP in SNU-407 and NCI-H508 cells (Fig. S4, A and B).

Figure 4. SPOP-mutant CRC cells are more sensitive to BETi and prone to DR5 induction.

Figure 4.

Open in a new tab

(A) MTS analysis of SPOP-WT (HCT116, RKO, SW48, Lim1215 and HT29) and SPOP-mutant (NCI-H508 and SNU-407) CRC cells treated with JQ1 or OTX015 at indicated concentrations for 72 hr. (B) Colony formation of the indicated CRC cells treated with JQ1 (0.5 μM) for 36 hr was analyzed by crystal violet staining. Upper, representative images of colonies; lower, enumeration of colony numbers. (C) Western blotting of BRD4 and SPOP in indicated SPOP-WT and -mutant CRC cell lines. (D) Upper, western blotting of DR5 in indicated SPOP-WT and -mutant CRC cell lines treated with JQ1 at indicated concentrations for 36 hr; lower, quantification of DR5 signals from both isoforms using the NIH Image J program, normalized to that of β-actin, and expressed as a ratio relative to untreated controls. (E) Western blotting of indicated proteins in NCI-H508 and SNU-407 cells transfected with control scrambled or SPOP siRNA and treated with JQ1 (0.5 μM) or OTX015 (0.5 μM) for 36 hr. Relative SPOP expression was quantified using Image J, normalized to that of β-actin, and expressed as a ratio relative to that in untreated cells that were transfected with control siRNA. (F) NCI-H508 and SNU-407 cells transfected with control scrambled or SPOP siRNA were treated with JQ1 (0.5 µM) or OTX015 (0.5 µM) for 36 hr. Apoptosis was analyzed by counting cells containing condensed and fragmented nuclei after nuclear staining. (G) MTS analysis of NCI-H508 and SNU-407 cells transfected with control scrambled or SPOP siRNA and treated with JQ1 or OTX015 at indicated concentrations for 72 hr. In (B) and (F), results were expressed as means ± SD of three independent experiments. *, P <0.05; **, P <0.01; ***, P <0.001.

Knockdown of DR5 by siRNA suppressed cell viability loss, nuclear fragmentation, and caspase activation induced by JQ1 and OTX015 in both SNU-407 and NCI-H508 cells (Fig. 5, A-C). Transfecting WT HCT116 cells with the activating mutants, including E47A or E50K (16), but not WT SPOP, increased JQ1 and OTX015 sensitivity (Fig. 5, D-F; Fig. S5A), which was accompanied by enhanced BRD4 depletion (Fig. 5D), apoptosis induction (Fig. 5G and S5B), and caspase activation (Fig. 5D and S5C). In contrast, these effects of SPOP mutants were almost completely absent in DR5-KO HCT116 cells (Fig. 5, E-H; Fig. S5, A-C). The DR5-dependent effects of SPOP mutants on BETi sensitivity were also observed in DR5-KO RKO cells transfected and treated in a similar fashion (Fig. S5, D-F). Together, these results indicate that increased BETi sensitivity in SPOP-mutant CRC cells is mediated by enhanced DR5 induction.

Figure 5. Increased BETi sensitivity of SPOP-mutant CRC cells is dependent on DR5.

Figure 5.

Open in a new tab

(A) Western blotting of indicated proteins in NCI-H508 and SNU-407 cells transfected with scrambled or DR5 siRNA and treated with JQ1 (0.5 μM) or OTX015 (0.5 μM) for 36 hr. Relative DR5 expression was quantified using the Image J program, normalized to that of β-actin, and expressed as a ratio relative to that in untreated cells that were transfected with control siRNA. (B) MTS analysis of NCI-H508 and SNU-407 cells transfected with control scrambled or DR5 siRNA and treated with JQ1 or OTX015 at indicated concentrations for 72 hr. (C) Apoptosis in NCI-H508 and SNU-407 cells transfected and treated as in (A) was analyzed by counting cells containing condensed and fragmented nuclei after nuclear staining. (D) Western blotting of HCT116 cells transfected with control empty vector, WT, E47A, or E50K mutant SPOP and treated with JQ1 (1 μM) for 36 hr. (E) MTS analysis of WT and DR5-KO HCT116 cells transfected with control empty vector, WT, E47A, or E50K mutant SPOP and treated with JQ1 at indicated concentrations for 72 hr. (F) Crystal violet staining of WT and DR5-KO HCT116 cells transfected as in (E) and treated with JQ1 (1 μM) for 36 hr. (G) Apoptosis in cells transfected and treated as in (F) were analyzed as in (C). (H) Western blotting of indicated proteins in cells transfected and treated as in (F). In (C) and (G), results were expressed as means ± SD of three independent experiments. *, P <0.05; **, P <0.01.

DR5 mediates the in vivo antitumor effects of JQ1/5-FU combination in cell line and PDX models.

To determine the role of DR5 in BETi chemosensitization in vivo, athymic nude mice with xenograft tumors established from WT and DR5-KO HCT116 cells were treated with 5-FU at a low, ineffective dose (i.p.; 25 mg/kg every other day), with or without JQ1 (i.p.; 30 mg/kg daily) (Fig. 6A). The combination treatment, but not JQ1 or 5-FU alone, effectively suppressed the growth of WT HCT116 tumors (Fig. 6, A and B). Compared to 5-FU or JQ1 alone, the combination treatment markedly enhanced DR5 induction (Fig. 6C) and activation of caspases 3 and 8 (Fig. 6, D-F). In contrast, the antitumor and apoptotic effects of the JQ1/5-FU combination treatment were largely suppressed in the DR5-KO tumors (Fig. 6, A-F).

Figure 6. DR5 mediates the chemosensitization effects of JQ1 in vivo.

Figure 6.

Open in a new tab

(A) Nude mice were injected subcutaneously with 4×106 WT or DR5-KO HCT116 cells. After tumor growth for 7 days, mice were treated with JQ1 (i.p.; 30 mg/kg daily), 5-FU (i.p.; 25 mg/kg every other day), or their combination as indicated for 3 weeks. Tumor volume at indicated time points after treatment was calculated and plotted with P values for indicated comparisons (n=6 in each group). (B) Representative tumors at the end of the experiment in (A). (C)-(F) Mice with WT and DR5-KO HCT116 xenograft tumors were treated as in (A) for 4 consecutive days. (C) Western blotting of DR5 in randomly selected WT tumors. Apoptosis was analyzed by (D) TUNEL, (E) active caspase 3, and (F) active caspase 8 staining of paraffin-embedded sections of WT or DR5-KO tumor tissues. Left, representative images with arrows indicating example positive signals (scale bars: 25 μm); right, quantification of positive signals. In (D)-(F), results were expressed as means ± SEM of analyzing 3 mice and at least 300 cells in each mouse. *, P <0.05; **, P <0.01.

PDX models better recapitulate heterogeneity, histology, and molecular alterations of patient tumors (36). We further explored the efficacy of the JQ1/5-FU combination in two CRC PDX models. The JQ1/5-FU combination, but not JQ1 (30 mg/kg daily) or 5-FU at a low dose (25 mg/kg every other day) alone, markedly suppressed PDX tumor growth (Fig. 7, A-D; Fig. S6A), without a significant effect on body weight (Fig. S6B). Compared to JQ1 or 5-FU alone, the combination treatment also induced higher levels of DR5 protein (Fig. 7E), tumor cell loss (Fig. 7F), suppression of cell proliferation detected by Ki67 staining (Fig. 7G), and apoptosis as analyzed by active caspase 3 and TUNEL staining (Fig. 7H; Fig. S6C). Collectively, our in vitro and in vivo data demonstrate a critical role of DR5 induction in mediating the chemosensitization and antitumor effects of BETi in CRC cells with different genetic backgrounds.

Figure 7. 5-FU/JQ1 combination suppresses the growth of PDX tumors.

Figure 7.

Open in a new tab

(A)-(D) NSG mice subcutaneously implanted with PDX1 or PDX2 model were treated with JQ1 (i.p.; 30 mg/kg daily), 5-FU (i.p.; 25 mg/kg every other day), or their combination for 20 days. (A) PDX1 tumor volume at indicated time points after treatment was calculated and plotted with P values for indicated comparisons (n=4 in each group). (B) Representative PDX1 tumors at the end of the experiment. (C) PDX2 tumor volume at indicated time points after treatment was calculated and plotted with P values for indicated comparisons (n=4 in each group). (D) Representative PDX2 tumors at the end of the experiment. (E)-(H) NSG mice bearing PDX1 or PDX2 tumors were treated as in (A) for 10 consecutive days. (E) Western blotting of DR5 in randomly selected tumors. Paraffin-embedded sections of PDX1 tumor tissues were analyzed by immunostaining for (F) H&E (scale bars: 100 μm), (G) Ki67, and (H) active caspase 3. In (F), the lower panels represent magnified sections of the areas indicated in the upper panels. In (G) and (H): left, representative images with arrows indicating example positive signals (scale bars: 25 μm); right, quantification of positive signals. Results were expressed as means ± SEM of analyzing 3 mice and at least 300 cells in each mouse. **, P <0.01; ***, P <0.001.

Discussion

We found that BET depletion or inhibition triggers ER stress and CHOP-mediated DR5 induction. Perturbations in ER function and ER stress can occur in normal cells in response to misfolded proteins or other stimuli (35). If unresolved, ER stress leads to eIF2α phosphorylation, CHOP and DR5 induction, and subsequent apoptotic death (37). This pathway can be engaged in cancer cells upon inhibition of addicted oncogenic signaling (38). ER stress and DR5 induction by BETi may result from downregulation of c-Myc, a key oncogenic driver activated by the defective APC/β-catenin pathway and aberrant Wnt signaling in CRC cells (7). Sustained c-Myc expression in tumor cells often relies on BET proteins, and is subject to inhibition by BETi (11). JQ1 has been shown to induce apoptosis in Eμ-Myc lymphoma cells (39). The extent of c-Myc depletion was found to correlate with BETi sensitivity in CRC cells (40). While directly targeting c-Myc is challenging, inhibiting its expression by BETi or other agents is an attractive approach for targeting Myc-driven cancers including CRCs. In line with our findings, other studies have also shown apoptosis induction as a key effect of BETi (19,20).

A combination of BETi with 5-FU or oxaliplatin markedly improved therapeutic efficacy in CRC cells, and may offer hope to patients with chemotherapy-refractory and metastatic CRCs (3). The improved therapeutic activity is dependent on enhanced DR5 induction, likely involving both p53-dependent and -independent mechanisms (22), leading to stronger apoptotic signaling via both the intrinsic and extrinsic pathways. The acute and potent induction of apoptosis by the combination treatment may provide a therapeutic window necessary for BETi dosing to minimize normal tissue toxicity caused by sustained BET inhibition, such as intestinal stem cell depletion and lymphoid and hematopoietic abnormalities (41,42). Our results showed that JQ1 at 30 mg/kg daily combined with 5-FU was well-tolerated and efficacious (Fig. 6 and 7). BETi were previously shown to synergize with other classes of anticancer agents including targeted drugs and immune checkpoint blockers (14). In those cases, BETi inhibited protective feedback mechanisms mediated by BRD-dependent upregulation of kinase signaling (14). Enhanced DR5 induction may contribute to improved therapeutic response via cell killing and subsequent effects on tumor microenvironment.

Several recent studies showed that mutations in SPOP, which encodes a substrate-binding subunit of a cullin-3-based E3 ubiquitin ligase, dictate BETi sensitivity in some cancer cells (15–17). Depending on tumor types and specific residues affected, SPOP mutations could be activating or inactivating, generating opposite effects on BETi sensitivity (15–17). Our results suggest that enhanced DR5 induction underlies the increased BETi sensitivity in SPOP-mutant CRC cells. The mutations we analyzed, including E47K in NCI-H508 and G75E in SNU-407, are located in the vicinity of other activating mutations, such as E47A, E50K, E78K, and S80R, which were mostly found in endometrial cancer cells (15–17). Our mechanistic data show that CRC cells expressing a SPOP-activating mutation have reduced BRD4 expression (Fig. 4C), and are more prone to DR5 induction in response to BETi (Fig. 4D). Analyzing additional SPOP mutations in different cancer types will be necessary in order to determine whether DR5 induction is broadly involved in modulating BETi sensitivity.

BETi have been evaluated in a number of clinical trials and shown single-agent efficacy against some hematologic malignancies and NUT carcinomas (14). Biomarkers for predicting BETi sensitivity are essential for developing personalized and precision therapies. In addition to SPOP mutations, CRC cells with a CpG island methylator phenotype (CIMP) are known to be sensitive to JQ1 and c-Myc repression (12). BETi resistance in triple-negative breast cancer cells involves hyper-phosphorylation and bromodomain-independent activities of BRD4 (43). Our findings suggest that ER stress and DR5 induction might be useful molecular markers for predicting BETi sensitivity. Several genes in the death receptor pathway, such as caspase 8, DCR1, and DCR3, are inactivated or downregulated in some CRCs (5). Our next step is to determine if DR5 or other genes in the death receptor pathway are aberrantly regulated in cells with acquired resistance to BETi due to changes in BRD4 and/or c-Myc.

In conclusion, we demonstrated a critical role of DR5-mediated apoptosis in determining the therapeutic response to BETi alone and BETi-mediated chemosensitization in CRC cells in vitro and in vivo. Our results provide a better understanding of the pro-apoptotic action of BETi, particularly the heightened sensitivity of SPOP-mutant CRCs.

Supplementary Material

1

Acknowledgments

We thank Dr. Darleny Y. Lizardo and other lab members for critical reading, Dr. Shi-Yong Sun at Emory University for providing the DR5 reporter constructs, and Dr. Zhanzhan Li at Xiangya Hospital of Central South University for help on statistical analyses of animal experiments. This work was supported by the U.S. National Institutes of Health grants (R01CA172136, R01CA213028, and R01CA217141 to L. Zhang; U19AI068021 and R01CA215481 to J. Yu; and U01CA152753 to R. E. Schoen) and UPMC Hillman Cancer Center institutional funds to L. Zhang. This project used the UPMC Hillman Cancer Center Animal Facility, Cytometry Facility, and Tissue and Research Pathology Services, which are supported in part by P30CA047904.

Footnotes

Conflict of interest: The authors have no conflict of interest.

References

  • 1.Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2017. CA Cancer J Clin 2017;67(1):7–30. [DOI] [PubMed] [Google Scholar]
  • 2.Chu E An update on the current and emerging targeted agents in metastatic colorectal cancer. Clinical colorectal cancer 2012;11(1):1–13. [DOI] [PubMed] [Google Scholar]
  • 3.Meyerhardt JA, Mayer RJ. Systemic therapy for colorectal cancer. N Engl J Med 2005;352(5):476–87. [DOI] [PubMed] [Google Scholar]
  • 4.Chen ML, Fang CH, Liang LS, Dai LH, Wang XK. A meta-analysis of chemotherapy regimen fluorouracil/leucovorin/oxaliplatin compared with fluorouracil/leucovorin in treating advanced colorectal cancer. Surgical oncology 2010;19(1):38–45. [DOI] [PubMed] [Google Scholar]
  • 5.Zhang L, Yu J. Role of apoptosis in colon cancer biology, therapy, and prevention. Curr Colorectal Cancer Rep 2013;9(4):331–40. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Le DT, Durham JN, Smith KN, Wang H, Bartlett BR, Aulakh LK, et al. Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade. Science 2017;357(6349):409–13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA Jr., Kinzler KW. Cancer genome landscapes. Science 2013;339(6127):1546–58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Feinberg AP, Koldobskiy MA, Gondor A. Epigenetic modulators, modifiers and mediators in cancer aetiology and progression. Nat Rev Genet 2016;17(5):284–99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Shi J, Vakoc CR. The mechanisms behind the therapeutic activity of BET bromodomain inhibition. Mol Cell 2014;54(5):728–36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Ferri E, Petosa C, McKenna CE. Bromodomains: Structure, function and pharmacology of inhibition. Biochem Pharmacol 2016;106:1–18. [DOI] [PubMed] [Google Scholar]
  • 11.Delmore JE, Issa GC, Lemieux ME, Rahl PB, Shi J, Jacobs HM, et al. BET bromodomain inhibition as a therapeutic strategy to target c-Myc. Cell 2011;146(6):904–17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.McCleland ML, Mesh K, Lorenzana E, Chopra VS, Segal E, Watanabe C, et al. CCAT1 is an enhancer-templated RNA that predicts BET sensitivity in colorectal cancer. J Clin Invest 2016;126(2):639–52. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Hu Y, Zhou J, Ye F, Xiong H, Peng L, Zheng Z, et al. BRD4 inhibitor inhibits colorectal cancer growth and metastasis. Int J Mol Sci 2015;16(1):1928–48. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Stathis A, Bertoni F. BET Proteins as Targets for Anticancer Treatment. Cancer discovery 2018;8(1):24–36. [DOI] [PubMed] [Google Scholar]
  • 15.Zhang P, Wang D, Zhao Y, Ren S, Gao K, Ye Z, et al. Intrinsic BET inhibitor resistance in SPOP-mutated prostate cancer is mediated by BET protein stabilization and AKT-mTORC1 activation. Nat Med 2017;23(9):1055–62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Janouskova H, El Tekle G, Bellini E, Udeshi ND, Rinaldi A, Ulbricht A, et al. Opposing effects of cancer-type-specific SPOP mutants on BET protein degradation and sensitivity to BET inhibitors. Nat Med 2017;23(9):1046–54. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Dai X, Gan W, Li X, Wang S, Zhang W, Huang L, et al. Prostate cancer-associated SPOP mutations confer resistance to BET inhibitors through stabilization of BRD4. Nat Med 2017;23(9):1063–71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100(1):57–70. [DOI] [PubMed] [Google Scholar]
  • 19.Conery AR, Centore RC, Spillane KL, Follmer NE, Bommi-Reddy A, Hatton C, et al. Preclinical Anticancer Efficacy of BET Bromodomain Inhibitors Is Determined by the Apoptotic Response. Cancer Res 2016;76(6):1313–9. [DOI] [PubMed] [Google Scholar]
  • 20.Yao W, Yue P, Khuri FR, Sun SY. The BET bromodomain inhibitor, JQ1, facilitates c-FLIP degradation and enhances TRAIL-induced apoptosis independent of BRD4 and c-Myc inhibition. Oncotarget 2015;6(33):34669–79. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Ashkenazi A Directing cancer cells to self-destruct with pro-apoptotic receptor agonists. Nat Rev Drug Discov 2008;7(12):1001–12. [DOI] [PubMed] [Google Scholar]
  • 22.Wu GS, Burns TF, McDonald ER 3rd, Jiang W, Meng R, Krantz ID, et al. KILLER/DR5 is a DNA damage-inducible p53-regulated death receptor gene. Nat Genet 1997;17(2):141–3. [DOI] [PubMed] [Google Scholar]
  • 23.He K, Zheng X, Li M, Zhang L, Yu J. mTOR inhibitors induce apoptosis in colon cancer cells via CHOP-dependent DR5 induction on 4E-BP1 dephosphorylation. Oncogene 2016;35(2):148–57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Moldoveanu T, Follis AV, Kriwacki RW, Green DR. Many players in BCL-2 family affairs. Trends in biochemical sciences 2014;39(3):101–11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Bhola PD, Letai A. Mitochondria-Judges and Executioners of Cell Death Sentences. Mol Cell 2016;61(5):695–704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Han B, Yao W, Oh YT, Tong JS, Li S, Deng J, et al. The novel proteasome inhibitor carfilzomib activates and enhances extrinsic apoptosis involving stabilization of death receptor 5. Oncotarget 2015;6(19):17532–42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Chen D, Tong J, Yang L, Wei L, Stolz DB, Yu J, et al. PUMA amplifies necroptosis signaling by activating cytosolic DNA sensors. Proc Natl Acad Sci U S A 2018;115(15):3930–35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Kim E, Ilic N, Shrestha Y, Zou L, Kamburov A, Zhu C, et al. Systematic Functional Interrogation of Rare Cancer Variants Identifies Oncogenic Alleles. Cancer discovery 2016;6(7):714–26. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Peng R, Tong JS, Li H, Yue B, Zou F, Yu J, et al. Targeting Bax interaction sites reveals that only homo-oligomerization sites are essential for its activation. Cell Death Differ 2013;20(5):744–54. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Wang P, Yu J, Zhang L. The nuclear function of p53 is required for PUMA-mediated apoptosis induced by DNA damage. Proc Natl Acad Sci U S A 2007;104(10):4054–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Yamaguchi H, Wang HG. CHOP is involved in endoplasmic reticulum stress-induced apoptosis by enhancing DR5 expression in human carcinoma cells. J Biol Chem 2004;279(44):45495–502. [DOI] [PubMed] [Google Scholar]
  • 32.Oh YT, Yue P, Zhou W, Balko JM, Black EP, Owonikoko TK, et al. Oncogenic Ras and B-Raf proteins positively regulate death receptor 5 expression through co-activation of ERK and JNK signaling. J Biol Chem 2012;287(1):257–67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Chen D, Wei L, Yu J, Zhang L. Regorafenib inhibits colorectal tumor growth through PUMA-mediated apoptosis. Clin Cancer Res 2014;20(13):3472–84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Li H, Wheeler S, Park Y, Ju Z, Thomas SM, Fichera M, et al. Proteomic Characterization of Head and Neck Cancer Patient-Derived Xenografts. Mol Cancer Res 2016;14(3):278–86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Tabas I, Ron D. Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress. Nat Cell Biol 2011;13(3):184–90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Jin K, Teng L, Shen Y, He K, Xu Z, Li G. Patient-derived human tumour tissue xenografts in immunodeficient mice: a systematic review. Clinical & translational oncology : official publication of the Federation of Spanish Oncology Societies and of the National Cancer Institute of Mexico 2010;12(7):473–80. [DOI] [PubMed] [Google Scholar]
  • 37.Lu M, Lawrence DA, Marsters S, Acosta-Alvear D, Kimmig P, Mendez AS, et al. Cell death. Opposing unfolded-protein-response signals converge on death receptor 5 to control apoptosis. Science 2014;345(6192):98–101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Li X, Li M, Ruan H, Qiu W, Xu X, Zhang L, et al. Co-targeting translation and proteasome rapidly kills colon cancer cells with mutant RAS/RAF via ER stress. Oncotarget 2017;8(6):9280–92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Hogg SJ, Newbold A, Vervoort SJ, Cluse LA, Martin BP, Gregory GP, et al. BET Inhibition Induces Apoptosis in Aggressive B-Cell Lymphoma via Epigenetic Regulation of BCL-2 Family Members. Mol Cancer Ther 2016;15(9):2030–41. [DOI] [PubMed] [Google Scholar]
  • 40.Togel L, Nightingale R, Chueh AC, Jayachandran A, Tran H, Phesse T, et al. Dual Targeting of Bromodomain and Extraterminal Domain Proteins, and WNT or MAPK Signaling, Inhibits c-MYC Expression and Proliferation of Colorectal Cancer Cells. Mol Cancer Ther 2016;15(6):1217–26. [DOI] [PubMed] [Google Scholar]
  • 41.Bolden JE, Tasdemir N, Dow LE, van Es JH, Wilkinson JE, Zhao Z, et al. Inducible In Vivo Silencing of Brd4 Identifies Potential Toxicities of Sustained BET Protein Inhibition. Cell Rep 2014. [DOI] [PMC free article] [PubMed]
  • 42.Lee DU, Katavolos P, Palanisamy G, Katewa A, Sioson C, Corpuz J, et al. Nonselective inhibition of the epigenetic transcriptional regulator BET induces marked lymphoid and hematopoietic toxicity in mice. Toxicology and applied pharmacology 2016;300:47–54. [DOI] [PubMed] [Google Scholar]
  • 43.Shu S, Lin CY, He HH, Witwicki RM, Tabassum DP, Roberts JM, et al. Response and resistance to BET bromodomain inhibitors in triple-negative breast cancer. Nature 2016;529(7586):413–17. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1519475-supplement-1.pdf (1.3MB, pdf)

RESOURCES