Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 May 6:5:10120.
doi: 10.1038/srep10120.

BET inhibitors induce apoptosis through a MYC independent mechanism and synergise with CDK inhibitors to kill osteosarcoma cells

Emma K Baker  1   2 , Scott Taylor  1 , Ankita Gupte  1 , Phillip P Sharp  3 , Mannu Walia  1 , Nicole C Walsh  1   2 , Andrew Cw Zannettino  4 , Alistair M Chalk  1   2 , Christopher J Burns  3   5 , Carl R Walkley  1   2   6
Affiliations

BET inhibitors induce apoptosis through a MYC independent mechanism and synergise with CDK inhibitors to kill osteosarcoma cells

Emma K Baker et al. Sci Rep. .

Abstract

Osteosarcoma (OS) survival rates have plateaued in part due to a lack of new therapeutic options. Here we demonstrate that bromodomain inhibitors (BETi), JQ1, I-BET151, I-BET762, exert potent anti-tumour activity against primary and established OS cell lines, mediated by inhibition of BRD4. Strikingly, unlike previous observations in long-term established human OS cell lines, the antiproliferative activity of JQ1 in primary OS cells was driven by the induction of apoptosis, not cell cycle arrest. In further contrast, JQ1 activity in OS was mediated independently of MYC downregulation. We identified that JQ1 suppresses the transcription factor FOSL1 by displacement of BRD4 from its locus. Loss of FOSL1 phenocopied the antiproliferative effects of JQ1, identifying FOSL1 suppression as a potential novel therapeutic approach for OS. As a monotherapy JQ1 demonstrated significant anti-tumour activity in vivo in an OS graft model. Further, combinatorial treatment approaches showed that JQ1 increased the sensitivity of OS cells to doxorubicin and induced potent synergistic activity when rationally combined with CDK inhibitors. The greater level of activity achieved with the combination of BETi with CDK inhibitors demonstrates the efficacy of this combination therapy. Taken together, our studies show that BET inhibitors are a promising new therapeutic for OS.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. BET inhibitors have potent activity against OS cell cultures
(a) IC50 values of OS cell cultures treated with JQ1 for 72 hrs. Mean IC50 values ± SEM (n = 2-4). (b) Dose response curves of mouse (494H, primary mouse fibroblastic; 148I, primary mouse osteoblastic) and human (OS17, primary human; MG63, established human OS cell line) cells treated with JQ1 or (-)-JQ1 for 72 hrs. Mean cell viability ± SEM (n = 3-4). (c) IC50 values of cells treated with I-BET151 and I-BET762 for 72 hrs. Mean IC50 values ± SEM (n = 2).
Figure 2
Figure 2. JQ1 induces apoptosis and cell cycle arrest in OS cell cultures
(a) Induction of cleaved caspase 3 in primary OS cell cultures (494H, primary mouse fibroblastic; 148I, primary mouse osteoblastic; OS17, primary human) treated with JQ1 for 24 hrs. (b) Annexin V and 7AAD staining of OS cells treated with 1 μM JQ1, 1 μM (-)-JQ1 or vehicle (DMSO) for 48 hrs. Plots are representative biological experiments with mean number of Annexin V positive cells (n = 3). (c) Cleaved caspase-3 levels in MG63 cells (established human OS cell line) treated with JQ1 for 24–48 hrs. (d) Annexin-V/7AAD staining of MG63 cells treated with 1 μM JQ1, 1 μM (-)-JQ1 or vehicle (DMSO) for 72 hrs. Plots are representative biological experiments with mean number of Annexin-V positive cells (n = 3). (e) Cell cycle profile of OS cells treated for 24 hrs with 1 μM JQ1, 1 μM (-)-JQ1 or vehicle (DMSO). Mean cell population percentage ± SEM (n = 4). (f) Dose response curves of mouse normal osteoblast cells treated with JQ1 or (-)-JQ1 for 72 hrs. Mean cell viability ± SEM (n = 3). (g) Cleaved caspase-3 levels in mouse normal osteoblast cells and mouse 494H cells treated with JQ1 for 24 hrs. (h) Annexin-V/ 7AAD staining of mouse normal osteoblast cells treated with 1 μM JQ1, 1 μM (-)-JQ1 or vehicle (DMSO) for 48 hrs. Plots are representative biological experiments with mean number of Annexin-V positive cells (n = 3).
Figure 3
Figure 3. JQ1 OS activity in vivo
Subcutaneous graft model of OS with 494H fibroblastic OS cells. (a) Body weight of mice undergoing treatment with JQ1 (50 mg/kg) or vehicle for 28 days. Mean weight ± SEM (n = 12). (b) Representative whole body bioluminescence images of mice at 28 days post treatment with JQ1 or vehicle. (c) Bioluminescence imaging assay of tumour burden in mice at day 0, 14, 21 and 28 post treatment with JQ1 or vehicle. Mean bioluminescence ± SEM (n = 12). Data was log transformed to normalise variances before analysis by 3-way ANOVA. (d) Representative microCT reconstructed images of tumours in mice treated for 28 days with JQ1 or vehicle. Tumour mass is circled in red. (e) MicroCT analysis of tumour bone volume in mice treated for 28 days with JQ1 or vehicle. Mean tumour bone volume ±SEM (n = 12). * p < 0.05 Mann Whitney ranks test. (f) Tumour weights from mice treated with JQ1 or vehicle for 28 days. Data is presented as mean ± SEM (n = 12, from two separate studies). * p < 0.05 calculated using Mann Whitney ranks test.
Figure 4
Figure 4. BRD4 knockdown phenocopies BET inhibitor activity in OS cells
(a) BRD4 transcript levels in mouse primary fibroblastic OS (494H, 493H, 716H, 202 V), mouse primary osteoblastic OS (148I, 98Sc, 89R, 147H) and human OS (U2OS, SJSA-1, B143, MG63, SAOS-2, G292, OS17) cells compared to normal osteoblast cells (n = 4-6 independently derived samples) quantified by QPCR. Mean relative expression ± SEM. *p < 0.05, ****p < 0.0001 ANOVA Tukey multiple comparisons test and student’s t test. (b-d) Human and mouse OS cells were transfected with siRNAs targeting BRD4 or a pool of non-targeting siRNAs (Non-T) and effects on BRD4 mRNA levels, protein expression and cell viability were assessed. (b) BRD4 transcript levels relative to Non-T cells quantified by QPCR. Mean fold change ± SEM (n = 3). ***p < 0.001, ****p < 0.0001 student’s t test.. (c) Western blot analyses showing BRD4 protein levels in siRNA transfected OS cells. (d) Cell viability in siRNA transfected OS cells. Cell viability levels are relative to Non-T cells. Mean fold change ± SEM (n = 3). *p < 0.05, ****p < 0.0001 student’s t test. (e) Western blot analyses of Brd4 and cleaved caspase 3 levels in 494H cells (primary mouse fibroblastic) or MG63 cells (established human OS cell line) transfected with siRNAs targeting BRD4 or a pool of non-targeting siRNAs (Non-T) for 72 hrs.
Figure 5
Figure 5. BET inhibitor activity in OS cells is driven by >FOSL1 suppression, independent of MYC downregulation
(a) Transcript levels of MYC, FOSL1 and RUNX2 were evaluated by QPCR in OS cells treated for 6 hrs or 24 hrs with 500 nM JQ1 or vehicle (DMSO). Mean fold change ± SEM (n = 3). **p < 0.01, ****p < 0.0001 ANOVA Dunnett multiple comparisons test. (b) Western blot analyses showing MYC and FOSL1 protein levels in OS cell cultures treated with JQ1 or vehicle (DMSO) for 24–48 hrs. Protein band intensities quantified by ImageJ. (c) FOSL1 transcript levels quantified by QPCR in OS cells transfected with siRNAs targeting FOSL1 or a pool of non-targeting siRNAs (Non-T) for 72 hrs. Mean fold change ± SEM (n = 3). **p < 0.01, ****p < 0.0001 student’s t test. (d) Cell viability in OS cells transfected with siRNAs targeting FOSL1 or a pool of non-targeting siRNAs (Non-T) for 72 hrs. Mean fold change ± SEM (n = 3). *p < 0.05, ***p < 0.001, ****p < 0.0001 student’s t test. (e) ChIP analysis of BRD4 enrichment at FOSL1, RUNX2 and MYC loci in human OS17 cells treated with 1 μM JQ1 or vehicle (DMSO) for 24 hrs. Enrichment is normalised to the total input DNA and a background region devoid of genes. Mean ± SEM (n = 3). * p < 0.05, ** p < 0.01 students t-test.
Figure 6
Figure 6. JQ1 enhances standard OS chemotherapy and synergistically combines with CDK inhibitors
(a) Dose response of OS cells treated with doxorubicin in the absence (columns) or presence (lines) of JQ1 for 72 hrs. Mean viability ± SEM (n = 3). (b) OS cells transfected with BRD4 siRNA or non-targeting siRNA (Non-T) and treated with 100 nM doxorubicin or vehicle (DMSO). Mean fold change cell viability ± SEM, (n = 3). ANOVA Tukey multiple comparisons test. (c) Cdk9 mRNA levels in mouse OS cells transfected with Cdk9 siRNA or Non-T siRNAs (Non-T) for 72 hrs. Mean fold change expression ± SEM (n = 3). ANOVA Dunnett multiple comparisons test. (d) Cell viability in mouse OS cells transfected with Cdk9 siRNA or Non-T siRNAs for 72 hrs. Mean fold change viability ± SEM (n = 3). ANOVA Dunnett multiple comparisons test. (e-f) Dose response curves of cells treated with flavopiridol or dinaciclib for 48 hrs. Mean ± SEM (n = 2). (g) AnnexinV/7AAD staining of OS17 cells treated with flavopiridol or vehicle (DMSO) for 24 hrs. Representative FACS plots; mean ± SEM (n = 4 DMSO, n = 3 Flavopiridol treated). ANOVA Dunnett multiple comparisons test. (h) OS cells treated with combinations of JQ1 with flavopiridol or dinaciclib for 72 hrs, represented as mean viability inhibition (n = 3). (i) Synergy effects of treatment with JQ1 combined with flavopiridol or dinaciclib for 72 hrs. Bliss additivity deviations greater than 15% were considered synergistic (red baseline), (n = 3). (j) Cell viability and synergy in OS cells transfected with BRD4 siRNA or Non-T siRNA and treated with 25 nM dinaciclib or vehicle (DMSO) for 72 hrs. Represented as mean cell viability normalised to vehicle treated Non-T siRNA ± SEM, (n = 3). Synergy is represented as mean percent deviation from a predicted additive response (Bliss additivity) (n = 3). (k) AnnexinV/7AAD staining of OS17 cells treated with combinations of JQ1 and dinaciclib for 48 hrs. Plots are representative biological experiments; results plotted as mean ± SEM Annexin-V positive cells (n = 4 per group). ANOVA Dunnett multiple comparisons test. For all panels: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bacci G. et al. Adjuvant and neoadjuvant chemotherapy for osteosarcoma of the extremities: 27 year experience at Rizzoli Institute, Italy. Eur. J. Cancer 41, 2836–2845 (2005). - PubMed
    1. Allison D. C. et al. A meta-analysis of osteosarcoma outcomes in the modern medical era. Sarcoma 2012, 704872 (2012). - PMC - PubMed
    1. Kaste S. C., Pratt C. B., Cain A. M., Jones-Wallace D. J. & Rao B. N. Metastases detected at the time of diagnosis of primary pediatric extremity osteosarcoma at diagnosis: imaging features. Cancer 86, 1602–1608 (1999). - PubMed
    1. Janeway K. A. & Grier H. E. Sequelae of osteosarcoma medical therapy: a review of rare acute toxicities and late effects. Lancet Oncol 11, 670–678 (2010). - PubMed
    1. Birch J. M. et al. Relative frequency and morphology of cancers in carriers of germline TP53 mutations. Oncogene 20, 4621–4628 (2001). - PubMed

Publication types

MeSH terms