Bromodomain and extraterminal 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 cells. Induction of DR5, following BET inhibition, was mediated by endoplasmic reticulum 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 colorectal cancer cells containing a mutation in speckle-type POZ protein (SPOP), a subunit of BRD4 E3 ubiquitin ligase. In a colorectal cancer xenograft model, BETi combined with chemotherapy suppressed the tumor growth in a DR5-dependent manner and potently inhibited patient-derived xenograft tumor growth with enhanced DR5 induction and apoptosis. These findings suggest that BETi alone or in combination with chemotherapy is effective against colorectal cancer due to enhanced DR5 induction and apoptosis. DR5 induction may also serve as a useful marker for designing personalized treatment and improved colorectal cancer combination therapies.

Significance: These 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.

Colorectal cancer is the second leading cause of cancer-related deaths in the United States (1). Patients with colorectal cancer are typically treated with conventional chemotherapeutic drugs including 5-fluorouracil (5-FU), oxaliplatin, and irinotecan (2), which have limited specificity and efficacy in advanced colorectal cancers (3, 4). Patients with colorectal cancer are also treated with targeted drugs such as the anti-EGFR antibodies, cetuximab and panitumumab, and the multikinase inhibitor, regorafenib (2). Nonetheless, the 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 colorectal cancers deficient in DNA mismatch repair (MMR; ref. 6). There is a critical need for developing novel therapies against colorectal cancers.

Epigenetic alterations such as DNA hypermethylation and histone modifications play an important role in colorectal cancer progression (7, 8). The bromodomain and extraterminal domain (BET) family proteins, including BRD2, BRD3, BRD4, and BRDT, are epigenetic readers of acetylated histone marks (9, 10). They function as transcriptional coactivators 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 colorectal cancer 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). Although 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 colorectal cancer cells. Our results suggest that DR5-mediated apoptosis plays a critical role in chemosensitization by BETi in colorectal cancer cells, and is responsible for increased BETi sensitivity in colorectal cancer cells with SPOP-activating mutations.

Cell culture and treatments

Human colorectal cancer cell lines, including HCT116, HT29, RKO, SW48, Lim1215, and NCI-H508, were purchased from the ATCC and cultured in McCoy 5A modified media (Invitrogen). SNU-407 was purchased from AddexBio and cultured in RPMI1640 (Invitrogen). DR5-knockout (KO) HCT116 cells were described previously (26). DR5-KO RKO cells were generated by CRISPR/Cas9 using a single guide RNA sequence 5′-CGCGGCGACAACGAGCACAA-3′ as described previously (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 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). For drug treatment, cells were plated in 12-well plates at 20%–30% density 24 hours 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 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 described previously (28), and obtained from Addgene. Small interfering RNA (siRNA) transfection was performed 24 hours before drug treatment using 200 pmol 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

Total RNA was prepared from HCT116 cells transfected with either control scrambled or BRD4 siRNA for 24 hours using the Quick-RNA Kit (Zymo Research) according to manufacturer's instructions. Library construction, RNA sequencing (RNA-seq), 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 on the basis of 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 described previously (29) using antibodies listed in Supplementary Table S1.

Real-time RT PCR

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

MTS assay

Cells seeded in 96-well plates at a density of 1 × 104 cells/well were treated with different agents for 72 hours. 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 described previously (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 described previously (33). All reporter experiments were performed in triplicate and repeated three times.

Chromatin immunoprecipitation

Chromatin immunoprecipitation (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 described previously (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; ref. 33). Viable cells in 12-well plates after drug treatment for 36 hours 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 tumor experiments

All animal experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Female 5- to 6-week-old Nu/Nu mice (Charles River) and 4- to 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: (i) untreated; (ii) JQ1 (i.p.; 30 mg/kg daily); (iii) 5-FU (i.p.; 25 mg/kg every other day); and (iv) 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 patient-derived xenograft (PDX) models were established using treatment-naïve primary tumors resected from patients with newly diagnosed colorectal cancers under written informed consent and approved Institutional Review Board 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 previously (34). Briefly, deidentified patient colorectal cancer samples were delivered to the laboratory in 1 × Antibiotic/Antimycotic Solution (Invitrogen) within 4 hours 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 described previously (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 t tests. Means ± SD are reported in the figures. For animal experiments, P values were calculated by repeated measures (RM) ANOVA with Fisher LSD post hoc tests. Means ± SEM are reported in the figures. Differences were considered significant if P < 0.05.

BETi induce DR5 expression through CHOP-mediated transcriptional activation

To determine the effects of BET inhibition in colorectal cancer 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. 1B and C). DR5 induction by BRD4 knockdown and BETi treatment was observed in other colorectal cancer cell lines with different colorectal cancer driver mutations (Supplementary Table S2), including those of KRAS, BRAF, PIK3CA, p53, SMAD4, SPOP, and FBW7, and associated with reduced c-Myc expression (Fig. 1D and E). The fold changes of both isoforms of DR5 protein were found to be higher than those of DR5 mRNA (Fig. 1A, C, and E), 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; Supplementary Fig. S1A). In contrast, the mRNA and protein expression of DR4 did not change significantly (Fig. 1A; Supplementary Fig. S1A–S1C). 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 colorectal cancer 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 colorectal cancer cells. A, HCT116 cells transfected with control scrambled or BRD4 siRNA were analyzed by RNA-seq at 24 hours 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 fragments per kilobase of transcript per million mapped reads (FPKM). B, Western blotting of DR5 in HCT116 cells treated with the BETi JQ1, I-BET151, OTX015, or I-BET762 at indicated concentrations for 36 hours. C, Western blot (top) and RT-PCR (bottom) analyses of DR5 expression at indicated time points in HCT116 cells treated with indicated BETi (1 μmol/L). D, Western blotting of indicated proteins in colorectal cancer cell lines with indicated p53, KRAS, BRAF, and PIK3CA status at 24 hours after transfection with control scrambled or BRD4 siRNA. E, Western blotting of indicated proteins (top) and RT-PCR of DR5 (bottom) in indicated colorectal cancer cell lines treated with JQ1 or OTX015 (1 μmol/L) for 36 hours. A–E, Relative expression of BRD4 and DR5 was quantified using the ImageJ 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 are expressed as means ± SD of three independent experiments (*, P < 0.05; **, P < 0.01).

Figure 1.

BETi treatment upregulates DR5 expression in colorectal cancer cells. A, HCT116 cells transfected with control scrambled or BRD4 siRNA were analyzed by RNA-seq at 24 hours 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 fragments per kilobase of transcript per million mapped reads (FPKM). B, Western blotting of DR5 in HCT116 cells treated with the BETi JQ1, I-BET151, OTX015, or I-BET762 at indicated concentrations for 36 hours. C, Western blot (top) and RT-PCR (bottom) analyses of DR5 expression at indicated time points in HCT116 cells treated with indicated BETi (1 μmol/L). D, Western blotting of indicated proteins in colorectal cancer cell lines with indicated p53, KRAS, BRAF, and PIK3CA status at 24 hours after transfection with control scrambled or BRD4 siRNA. E, Western blotting of indicated proteins (top) and RT-PCR of DR5 (bottom) in indicated colorectal cancer cell lines treated with JQ1 or OTX015 (1 μmol/L) for 36 hours. A–E, Relative expression of BRD4 and DR5 was quantified using the ImageJ 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 are expressed as means ± SD of three independent experiments (*, P < 0.05; **, P < 0.01).

Close modal

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 and E; Supplementary Fig. S1D). RNA-seq data revealed upregulation of several ER stress response genes including CHOP following BRD4 depletion (Supplementary Fig. S1E), which was confirmed by protein expression (Fig. 2A). Treating colorectal cancer cells with different BETi also induced CHOP mRNA and protein (Fig. 2B; Supplementary Fig. S1F), as well as other ER stress markers including BiP expression and eIF2α (Ser51) and PERK (Thr980) phosphorylation (Fig. 2B; ref. 35). Knockdown of CHOP by siRNA abrogated the induction of DR5 mRNA and protein by JQ1 (Fig. 2C). 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 (Fig. 2E; ref. 32). Together, these results indicate that BETi treatment induces DR5 through ER stress/CHOP-mediated transcription.

Figure 2.

BETi induces DR5 expression through ER stress and CHOP-mediated transcription. A, Western blot (left) and RT-PCR (right) analyses of DR5 and CHOP in HCT116 cells at 24 hours after transfection with control scrambled or BRD4 siRNA. B, Western blotting of CHOP and indicated ER stress markers (top) and RT-PCR (bottom) of CHOP in indicated colorectal cancer cell lines treated with indicated BETi (1 μmol/L) for 36 hours. p-eIF2α, phospho-eIF2α; Ser51; p-PERK, phospho-PERK, Thr980. C, Western blotting of CHOP and DR5 (top) and RT-PCR (bottom) of DR5 in HCT116 and RKO cells transfected with control scrambled or CHOP siRNA and treated with JQ1 (1 μmol/L) for 36 hours. D, ChIP analysis of the binding of CHOP to the DR5 promoter in HCT116 cells treated with JQ1 (1 μmol/L) for 36 hours, 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 WT or mutant CHOP-binding site, along with the transfection control β-galactosidase reporter pCMVβ. After 24 hours, cells were treated with JQ1 (1 μmol/L) for 36 hours. Top, a schematic representation of the CHOP-binding site in the DR5 promoter, with “xxxx” indicating mutated nucleotides in the mutant reporter; bottom, 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 ImageJ, 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 are expressed as means ± SD of three independent experiments (*, P < 0.05; **, P < 0.01).

Figure 2.

BETi induces DR5 expression through ER stress and CHOP-mediated transcription. A, Western blot (left) and RT-PCR (right) analyses of DR5 and CHOP in HCT116 cells at 24 hours after transfection with control scrambled or BRD4 siRNA. B, Western blotting of CHOP and indicated ER stress markers (top) and RT-PCR (bottom) of CHOP in indicated colorectal cancer cell lines treated with indicated BETi (1 μmol/L) for 36 hours. p-eIF2α, phospho-eIF2α; Ser51; p-PERK, phospho-PERK, Thr980. C, Western blotting of CHOP and DR5 (top) and RT-PCR (bottom) of DR5 in HCT116 and RKO cells transfected with control scrambled or CHOP siRNA and treated with JQ1 (1 μmol/L) for 36 hours. D, ChIP analysis of the binding of CHOP to the DR5 promoter in HCT116 cells treated with JQ1 (1 μmol/L) for 36 hours, 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 WT or mutant CHOP-binding site, along with the transfection control β-galactosidase reporter pCMVβ. After 24 hours, cells were treated with JQ1 (1 μmol/L) for 36 hours. Top, a schematic representation of the CHOP-binding site in the DR5 promoter, with “xxxx” indicating mutated nucleotides in the mutant reporter; bottom, 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 ImageJ, 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 are expressed as means ± SD of three independent experiments (*, P < 0.05; **, P < 0.01).

Close modal

Chemosensitization by BETi requires DR5-mediated apoptosis in colorectal cancer cells

BRD4 knockdown or inhibition, while ineffective alone (Supplementary Fig. S2A and S2B), markedly suppressed colorectal cancer cell growth when combined with 5-FU or oxaliplatin (Fig. 3A and B; Supplementary Fig. S2B and S2C). 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 (Supplementary Fig. S2C–S2G). Enhanced apoptosis by the 5-FU/BETi combination was also observed in RKO cells (Supplementary 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. 3C–E; Supplementary Fig. S3A–S3C), and significantly improved the long-term clonogenic survival (Fig. 3F). Furthermore, KO of DR5 by CRISPR/Cas9 in RKO cells also abolished apoptosis induced by BRD4 knockdown or JQ1 in combination with 5-FU or oxaliplatin (Supplementary Fig. S3D and S3E). Together, these results demonstrate that the chemosensitization effects of BETi are dependent on DR5-mediated apoptosis in colorectal cancer cells.

Figure 3.

Chemosensitization by BETi is dependent on DR5-mediated apoptosis. 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 hours. B, MTS analysis of HCT116 cells treated with 5-FU or OXA at indicated concentrations with or without JQ1 (1 μmol/L) or OTX015 (1 μmol/L) for 72 hours. 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 μmol/L) for 36 hours with or without BRD4 knockdown as in A or BETi treatment as in B. Apoptosis was analyzed by counting the 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 ImageJ 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. Top, representative images of colonies; bottom, enumeration of colony numbers. In D and F, results are expressed as means ± SD of three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 3.

Chemosensitization by BETi is dependent on DR5-mediated apoptosis. 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 hours. B, MTS analysis of HCT116 cells treated with 5-FU or OXA at indicated concentrations with or without JQ1 (1 μmol/L) or OTX015 (1 μmol/L) for 72 hours. 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 μmol/L) for 36 hours with or without BRD4 knockdown as in A or BETi treatment as in B. Apoptosis was analyzed by counting the 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 ImageJ 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. Top, representative images of colonies; bottom, enumeration of colony numbers. In D and F, results are expressed as means ± SD of three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Close modal

SPOP-mutant colorectal cancer cells are exquisitely sensitive to BETi due to enhanced DR5 induction

Recent studies showed that the 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 (Supplementary Table S2), we identified another colorectal cancer line, SNU-407, which contains a SPOP mutation (G75E), in addition to NCI-H508 (SPOP-E47K; ref. 16). SNU-407 and NCI-H508 cells were more sensitive to BETi than SPOP-WT colorectal cancer cells lines, with IC50 values of JQ1 and OXT015 ranging from 0.69 to 1.05 μmol/L, compared with 4.26 to 12.02 μmol/L in HCT116, HT29, RKO, SW48, and Lim1205 cells (Fig. 4A). JQ1 at 0.5 μmol/L potently suppressed colony formation of SNU-407 and NCI-H508 cells but barely affected that of HCT116 cells (Fig. 4B). Compared with SPOP-WT colorectal cancer 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 μmol/L, in contrast to an obvious DR5 induction with JQ1 at 4 μmol/L in the SPOP-WT cells (Fig. 4D). Consistent with the strong DR5 induction, robust activation of caspases 3 and 8 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, caspase-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 (Supplementary Fig. S4A and S4B).

Figure 4.

SPOP-mutant colorectal cancer cells are more sensitive to BETi and prone to DR5 induction. A, MTS analysis of SPOP-WT (HCT116, RKO, SW48, Lim1215, and HT29) and SPOP-mutant (NCI-H508 and SNU-407) colorectal cancer cells treated with JQ1 or OTX015 at indicated concentrations for 72 hours. B, Colony formation of the indicated colorectal cancer cells treated with JQ1 (0.5 μmol/L) for 36 hours was analyzed by crystal violet staining 14 days after treatment. Top, representative images of colonies; bottom, enumeration of colony numbers. C, Western blotting of BRD4 and SPOP in indicated SPOP-WT and -mutant colorectal cancer cell lines. D, Top, Western blotting of DR5 in indicated SPOP-WT and -mutant colorectal cancer cell lines treated with JQ1 at indicated concentrations for 36 hours; bottom, quantification of DR5 signals from both isoforms using the NIH ImageJ 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 μmol/L) or OTX015 (0.5 μmol/L) for 36 hours. Relative SPOP expression was quantified using ImageJ, 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 μmol/L) or OTX015 (0.5 μmol/L) for 36 hours. 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 hours. In B and F, results are expressed as means ± SD of three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 4.

SPOP-mutant colorectal cancer cells are more sensitive to BETi and prone to DR5 induction. A, MTS analysis of SPOP-WT (HCT116, RKO, SW48, Lim1215, and HT29) and SPOP-mutant (NCI-H508 and SNU-407) colorectal cancer cells treated with JQ1 or OTX015 at indicated concentrations for 72 hours. B, Colony formation of the indicated colorectal cancer cells treated with JQ1 (0.5 μmol/L) for 36 hours was analyzed by crystal violet staining 14 days after treatment. Top, representative images of colonies; bottom, enumeration of colony numbers. C, Western blotting of BRD4 and SPOP in indicated SPOP-WT and -mutant colorectal cancer cell lines. D, Top, Western blotting of DR5 in indicated SPOP-WT and -mutant colorectal cancer cell lines treated with JQ1 at indicated concentrations for 36 hours; bottom, quantification of DR5 signals from both isoforms using the NIH ImageJ 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 μmol/L) or OTX015 (0.5 μmol/L) for 36 hours. Relative SPOP expression was quantified using ImageJ, 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 μmol/L) or OTX015 (0.5 μmol/L) for 36 hours. 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 hours. In B and F, results are expressed as means ± SD of three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Close modal

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. 5A–C). Transfecting WT HCT116 cells with the activating mutants, including E47A or E50K (16), but not WT SPOP, increased JQ1 and OTX015 sensitivity (Fig. 5D–F; Supplementary Fig. S5A), which was accompanied by enhanced BRD4 depletion (Fig. 5D), apoptosis induction (Fig. 5G; Supplementary Fig. S5B), and caspase activation (Fig. 5D; Supplementary Fig. S5C). In contrast, these effects of SPOP mutants were almost completely absent in DR5-KO HCT116 cells (Fig. 5E–H; Supplementary Fig. S5A–S5C). 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 (Supplementary Fig. S5D–S5F). Together, these results indicate that increased BETi sensitivity in SPOP-mutant colorectal cancer cells is mediated by enhanced DR5 induction.

Figure 5.

Increased BETi sensitivity of SPOP-mutant colorectal cancer cells is dependent on DR5. 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 μmol/L) or OTX015 (0.5 μmol/L) for 36 hours. 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 hours. 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 (0.5 μmol/L) for 36 hours. 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 hours. F, Crystal violet staining of WT and DR5-KO HCT116 cells transfected as in E and treated with JQ1 (0.5 μmol/L) for 36 hours. 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 are expressed as means ± SD of three independent experiments (*, P < 0.05; **, P < 0.01).

Figure 5.

Increased BETi sensitivity of SPOP-mutant colorectal cancer cells is dependent on DR5. 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 μmol/L) or OTX015 (0.5 μmol/L) for 36 hours. 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 hours. 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 (0.5 μmol/L) for 36 hours. 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 hours. F, Crystal violet staining of WT and DR5-KO HCT116 cells transfected as in E and treated with JQ1 (0.5 μmol/L) for 36 hours. 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 are expressed as means ± SD of three independent experiments (*, P < 0.05; **, P < 0.01).

Close modal

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. 6A and B). Compared with 5-FU or JQ1 alone, the combination treatment markedly enhanced DR5 induction (Fig. 6C) and activation of caspases 3 and 8 (Fig. 6D–F). In contrast, the antitumor and apoptotic effects of the JQ1/5-FU combination treatment were largely suppressed in the DR5-KO tumors (Fig. 6A–F).

Figure 6.

DR5 mediates the chemosensitization effects of JQ1 in vivo. 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 TUNEL (D), active caspase-3 (E), and active caspase-8 (F) staining of paraffin-embedded sections of WT or DR5-KO tumor tissues. Left, representative images with arrows indicating example of positive signals (scale bars, 25 μm); right, quantification of positive signals. DF, Results are expressed as means ± SEM of analyzing three mice and at least 300 cells in each mouse (*, P < 0.05; **, P < 0.01).

Figure 6.

DR5 mediates the chemosensitization effects of JQ1 in vivo. 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 TUNEL (D), active caspase-3 (E), and active caspase-8 (F) staining of paraffin-embedded sections of WT or DR5-KO tumor tissues. Left, representative images with arrows indicating example of positive signals (scale bars, 25 μm); right, quantification of positive signals. DF, Results are expressed as means ± SEM of analyzing three mice and at least 300 cells in each mouse (*, P < 0.05; **, P < 0.01).

Close modal

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 colorectal cancer 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. 7A–D; Supplementary Fig. S6A), without a significant effect on body weight (Supplementary Fig. S6B). Compared with 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; Supplementary 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 colorectal cancer cells with different genetic backgrounds.

Figure 7.

5-FU/JQ1 combination suppresses the growth of PDX tumors. 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. EH, 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 hematoxylin and eosin (scale bars, 100 μm; F), Ki67 (G), and active caspase-3 (H). F, The bottom panels represent magnified sections of the areas indicated in the top panels. G and H, Left, representative images with arrows indicating example positive signals (scale bars, 25 μm); right, quantification of positive signals. Results are expressed as means ± SEM of analyzing three mice and at least 300 cells in each mouse (**, P < 0.01; ***, P < 0.001).

Figure 7.

5-FU/JQ1 combination suppresses the growth of PDX tumors. 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. EH, 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 hematoxylin and eosin (scale bars, 100 μm; F), Ki67 (G), and active caspase-3 (H). F, The bottom panels represent magnified sections of the areas indicated in the top panels. G and H, Left, representative images with arrows indicating example positive signals (scale bars, 25 μm); right, quantification of positive signals. Results are expressed as means ± SEM of analyzing three mice and at least 300 cells in each mouse (**, P < 0.01; ***, P < 0.001).

Close modal

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 colorectal cancer 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 colorectal cancer cells (40). Although directly targeting c-Myc is challenging, inhibiting its expression by BETi or other agents is an attractive approach for targeting Myc-driven cancers including colorectal cancers. 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 colorectal cancer cells, and may offer hope to patients with chemotherapy-refractory and metastatic colorectal cancers (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 the 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 the 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 colorectal cancer 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 colorectal cancer 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 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, colorectal cancer 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 hyperphosphorylation 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 colorectal cancers (5). Our next step is to determine whether 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 colorectal cancer cells in vitro and in vivo. Our results provide a better understanding of the proapoptotic action of BETi, particularly the heightened sensitivity of SPOP-mutant colorectal cancers.

No potential conflicts of interest were disclosed.

Conception and design: X. Tan, L. Zhang

Development of methodology: R. Fletcher, L. Zhang

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): X. Tan, J. Tong, Y.-J. Wang, R. Fletcher, J. Yu, L. Zhang, R.E. Schoen

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): X. Tan, J. Tong, Y.-J. Wang, L. Zhang

Writing, review, and/or revision of the manuscript: X. Tan, Y.-J. Wang, R. Fletcher, J. Yu, L. Shen, L. Zhang, R.E. Schoen

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Yu, L. Shen, L. Zhang

Study supervision: L. Shen, L. Zhang

We thank Dr. Darleny Y. Lizardo and other lab members for critical reading, Dr. Shi-Yong Sun at Emory University (Atlanta, GA) for providing the DR5 reporter constructs, and Dr. Zhanzhan Li at Xiangya Hospital of Central South University (Changsha, Hunan, China) for help on statistical analyses of animal experiments. This work was supported by the NIH 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.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Siegel
RL
,
Miller
KD
,
Jemal
A
. 
Cancer statistics, 2017
.
CA Cancer J Clin
2017
;
67
:
7
30
.
2.
Chu
E
. 
An update on the current and emerging targeted agents in metastatic colorectal cancer
.
Clin Colorect Cancer
2012
;
11
:
1
13
.
3.
Meyerhardt
JA
,
Mayer
RJ
. 
Systemic therapy for colorectal cancer
.
N Engl J Med
2005
;
352
:
476
87
.
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
.
Surg Oncol
2010
;
19
:
38
45
.
5.
Zhang
L
,
Yu
J
. 
Role of apoptosis in colon cancer biology, therapy, and prevention
.
Curr Colorectal Cancer Rep
2013
;
9
:
331
40
.
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
:
409
13
.
7.
Vogelstein
B
,
Papadopoulos
N
,
Velculescu
VE
,
Zhou
S
,
Diaz
LA
 Jr.
,
Kinzler
KW
. 
Cancer genome landscapes
.
Science
2013
;
339
:
1546
58
.
8.
Feinberg
AP
,
Koldobskiy
MA
,
Gondor
A
. 
Epigenetic modulators, modifiers and mediators in cancer aetiology and progression
.
Nat Rev Genet
2016
;
17
:
284
99
.
9.
Shi
J
,
Vakoc
CR
. 
The mechanisms behind the therapeutic activity of BET bromodomain inhibition
.
Mol Cell
2014
;
54
:
728
36
.
10.
Ferri
E
,
Petosa
C
,
McKenna
CE
. 
Bromodomains: structure, function and pharmacology of inhibition
.
Biochem Pharmacol
2016
;
106
:
1
18
.
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
:
904
17
.
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
:
639
52
.
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
:
1928
48
.
14.
Stathis
A
,
Bertoni
F
. 
BET proteins as targets for anticancer treatment
.
Cancer Discov
2018
;
8
:
24
36
.
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
:
1055
62
.
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
:
1046
54
.
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
:
1063
71
.
18.
Hanahan
D
,
Weinberg
RA
. 
The hallmarks of cancer
.
Cell
2000
;
100
:
57
70
.
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
:
1313
9
.
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
:
34669
79
.
21.
Ashkenazi
A
. 
Directing cancer cells to self-destruct with pro-apoptotic receptor agonists
.
Nat Rev Drug Discov
2008
;
7
:
1001
12
.
22.
Wu
GS
,
Burns
TF
,
McDonald
ER
 III
,
Jiang
W
,
Meng
R
,
Krantz
ID
, et al
KILLER/DR5 is a DNA damage-inducible p53-regulated death receptor gene
.
Nat Genet
1997
;
17
:
141
3
.
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
:
148
57
.
24.
Moldoveanu
T
,
Follis
AV
,
Kriwacki
RW
,
Green
DR
. 
Many players in BCL-2 family affairs
.
Trends Biochem Sci
2014
;
39
:
101
11
.
25.
Bhola
PD
,
Letai
A
. 
Mitochondria-judges and executioners of cell death sentences
.
Mol Cell
2016
;
61
:
695
704
.
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
:
17532
42
.
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
:
3930
35
.
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 Discov
2016
;
6
:
714
26
.
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
:
744
54
.
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
:
4054
9
.
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
:
45495
502
.
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
:
257
67
.
33.
Chen
D
,
Wei
L
,
Yu
J
,
Zhang
L
. 
Regorafenib inhibits colorectal tumor growth through PUMA-mediated apoptosis
.
Clin Cancer Res
2014
;
20
:
3472
84
.
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
:
278
86
.
35.
Tabas
I
,
Ron
D
. 
Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress
.
Nat Cell Biol
2011
;
13
:
184
90
.
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
.
Clin Translat Oncol
2010
;
12
:
473
80
.
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
:
98
101
.
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
:
9280
92
.
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
:
2030
41
.
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
:
1217
26
.
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
;
8
:
1919
29
.
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
.
Toxicol Appl Pharmacol
2016
;
300
:
47
54
.
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
:
413
17
.