Bromodomain Inhibitor JQ1 Provides Novel Insights and Perspectives in Rhabdomyosarcoma Treatment
Abstract
:1. Introduction
2. Results
2.1. Expression of MYC Is Variable between RMS Cell Lines and Does Not Depend on the Fusion State of PAX3-FOXO1
2.2. JQ1 Sensitivity Is Associated with MYC Steady-State Levels
2.3. JQ1 Induce Cell Growth Arrest and Cell Death in RMS 3D Tumor Spheroids
2.4. JQ1 Sensitivity Is Associated to Cell Cycle Arrest and Apoptosis
3. Discussions
4. Materials and Methods
4.1. Cell Culture
4.2. Kinetics of Cytotoxicity and Cell Proliferation
4.3. Kinetics of 3D Tumor Spheroid Growth and Survival
4.4. Cell Cycle Analysis
4.5. Western Blot
4.6. Datasets
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Ognjanovic, S.; Linabery, A.M.; Charbonneau, B.; Ross, J.A. Trends in childhood rhabdomyosarcoma incidence and survival in the United States, 1975–2005. Cancer 2009, 115, 4218–4226. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Dasgupta, R.; Fuchs, J.; Rodeberg, D. Rhabdomyosarcoma. Semin. Pediatr. Surg. 2016, 25, 276–283. [Google Scholar] [CrossRef] [PubMed]
- Saab, R.; Spunt, S.L.; Skapek, S.X. Myogenesis and rhabdomyosarcoma the Jekyll and Hyde of skeletal muscle. Curr. Top. Dev. Biol. 2011, 94, 197–234. [Google Scholar] [CrossRef] [PubMed]
- Marchesi, I.; Giordano, A.; Bagella, L. Roles of enhancer of zeste homolog 2: From skeletal muscle differentiation to rhabdomyosarcoma carcinogenesis. Cell Cycle 2014, 13, 516–527. [Google Scholar] [CrossRef] [Green Version]
- Zoroddu, S.; Marchesi, I.; Bagella, L. PRC2: An epigenetic multiprotein complex with a key role in the development of rhabdomyosarcoma carcinogenesis. Clin. Epigenetics 2021, 13, 156. [Google Scholar] [CrossRef]
- Smith, M.A.; Altekruse, S.F.; Adamson, P.C.; Reaman, G.H.; Seibel, N.L. Declining childhood and adolescent cancer mortality. Cancer 2014, 120, 2497–2506. [Google Scholar] [CrossRef]
- Mazzoleni, S.; Bisogno, G.; Garaventa, A.; Cecchetto, G.; Ferrari, A.; Sotti, G.; Donfrancesco, A.; Madon, E.; Casula, L.; Carli, M. Outcomes and prognostic factors after recurrence in children and adolescents with nonmetastatic rhabdomyosarcoma. Cancer 2005, 104, 183–190. [Google Scholar] [CrossRef]
- Oberlin, O.; Rey, A.; Lyden, E.; Bisogno, G.; Stevens, M.C.G.; Meyer, W.H.; Carli, M.; Anderson, J.R. Prognostic factors in metastatic rhabdomyosarcomas: Results of a pooled analysis from United States and European cooperative groups. J. Clin. Oncol. 2008, 26, 2384–2389. [Google Scholar] [CrossRef] [Green Version]
- Toffolatti, L.; Frascella, E.; Ninfo, V.; Gambini, C.; Forni, M.; Carli, M.; Rosolen, A. MYCN expression in human rhabdomyosarcoma cell lines and tumour samples. J. Pathol. 2002, 196, 450–458. [Google Scholar] [CrossRef]
- Marampon, F.; Ciccarelli, C.; Zani, B.M. Down-regulation of c-Myc following MEK/ERK inhibition halts the expression of malignant phenotype in rhabdomyosarcoma and in non muscle-derived human tumors. Mol. Cancer 2006, 5. [Google Scholar] [CrossRef] [Green Version]
- Tonelli, R.; McIntyre, A.; Camerin, C.; Walters, Z.S.; di Leo, K.; Selfe, J.; Purgato, S.; Missiaglia, E.; Tortori, A.; Renshaw, J.; et al. Antitumor Activity of Sustained N-Myc Reduction in Rhabdomyosarcomas and Transcriptional Block by Antigene Therapy. Clin. Cancer Res. 2012, 18, 796–807. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Zhang, J.; Song, N.; Zang, D.; Yu, J.; Li, J.; Di, W.; Guo, R.; Zhao, W.; Wang, H. c-Myc promotes tumor proliferation and anti-apoptosis by repressing p21 in rhabdomyosarcomas. Mol. Med. Rep. 2017, 16, 4089–4094. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Shi, J.; Vakoc, C.R. The Mechanisms behind the Therapeutic Activity of BET Bromodomain Inhibition. Mol. Cell 2014, 54, 728–736. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Fu, L.-L.; Tian, M.; Li, X.; Li, J.-J.; Huang, J.; Ouyang, L.; Zhang, Y.; Liu, B. Inhibition of BET bromodomains as a therapeutic strategy for cancer drug discovery. Oncotarget 2015, 6, 5501–5516. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Kato, F.; Fiorentino, F.P.; Alibés, A.; Perucho, M.; Sánchez-Céspedes, M.; Kohno, T.; Yokota, J. MYCL is a target of a BET bromodomain inhibitor, JQ1, on growth suppression efficacy in small cell lung cancer cells. Oncotarget 2016, 7, 77378–77388. [Google Scholar] [CrossRef] [Green Version]
- Fiorentino, F.P.; Marchesi, I.; Schröder, C.; Schmidt, R.; Yokota, J.; Bagella, L. BET-Inhibitor I-BET762 and PARP-Inhibitor Talazoparib Synergy in Small Cell Lung Cancer Cells. Int. J. Mol. Sci. 2020, 21, 9595. [Google Scholar] [CrossRef]
- Kanno, T.; Kanno, Y.; LeRoy, G.; Campos, E.; Sun, H.-W.; Brooks, S.R.; Vahedi, G.; Heightman, T.D.; Garcia, B.A.; Reinberg, D.; et al. BRD4 assists elongation of both coding and enhancer RNAs by interacting with acetylated histones. Nat. Struct. Mol. Biol. 2014, 21, 1047–1057. [Google Scholar] [CrossRef] [Green Version]
- Lovén, J.; Hoke, H.A.; Lin, C.Y.; Lau, A.; Orlando, D.A.; Vakoc, C.R.; Bradner, J.E.; Lee, T.I.; Young, R.A. Selective Inhibition of Tumor Oncogenes by Disruption of Super-Enhancers. Cell 2013, 153, 320–334. [Google Scholar] [CrossRef] [Green Version]
- Bid, H.K.; Phelps, D.A.; Xaio, L.; Guttridge, D.C.; Lin, J.; London, C.; Baker, L.H.; Mo, X.; Houghton, P.J. The Bromodomain BET Inhibitor JQ1 Suppresses Tumor Angiogenesis in Models of Childhood Sarcoma. Mol. Cancer Ther. 2016, 15, 1018–1028. [Google Scholar] [CrossRef] [Green Version]
- Gryder, B.E.; Yohe, M.E.; Chou, H.-C.; Zhang, X.; Marques, J.; Wachtel, M.; Schaefer, B.; Sen, N.; Song, Y.; Gualtieri, A.; et al. PAX3–FOXO1 Establishes Myogenic Super Enhancers and Confers BET Bromodomain Vulnerability. Cancer Discov. 2017, 7, 884–899. [Google Scholar] [CrossRef] [Green Version]
- Fiorentino, F.P.; Tokgün, E.; Solé-Sánchez, S.; Giampaolo, S.; Tokgün, O.; Jauset, T.; Kohno, T.; Perucho, M.; Soucek, L.; Yokota, J. Growth suppression by MYC inhibition in small cell lung cancer cells with TP53 and RB1 inactivation. Oncotarget 2016, 7, 31014–31028. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Fiorentino, F.P.; Bagella, L.; Marchesi, I. A new parameter of growth inhibition for cell proliferation assays. J. Cell. Physiol. 2018, 233, 4106–4115. [Google Scholar] [CrossRef] [PubMed]
- Selby, M.; Delosh, R.; Laudeman, J.; Ogle, C.; Reinhart, R.; Silvers, T.; Lawrence, S.; Kinders, R.; Parchment, R.; Teicher, B.A.; et al. 3D Models of the NCI60 Cell Lines for Screening Oncology Compounds. SLAS Discov. Adv. Sci. Drug Discov. 2017, 22, 473–483. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Dawson, M.A.; Kouzarides, T. Cancer Epigenetics: From Mechanism to Therapy. Cell 2012, 150, 12–27. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Cote, G.M.; Choy, E. Role of Epigenetic Modulation for the Treatment of Sarcoma. Curr. Treat. Options Oncol. 2013, 14, 454–464. [Google Scholar] [CrossRef] [PubMed]
- Pal, A.; Chiu, H.Y.; Taneja, R. Genetics, epigenetics and redox homeostasis in rhabdomyosarcoma: Emerging targets and therapeutics. Redox Biol. 2019, 25, 101124. [Google Scholar] [CrossRef]
- Kutko, M.C.; Glick, R.D.; Butler, L.M.; Coffey, D.C.; Rifkind, R.A.; Marks, P.A.; Richon, V.M.; LaQuaglia, M.P. Histone deacetylase inhibitors induce growth suppression and cell death in human rhabdomyosarcoma in vitro. Clin. Cancer Res. 2003, 9, 5749–5755. [Google Scholar]
- Hedrick, E.; Crose, L.; Linardic, C.M.; Safe, S. Histone Deacetylase Inhibitors Inhibit Rhabdomyosarcoma by Reactive Oxygen Species-Dependent Targeting of Specificity Protein Transcription Factors. Mol. Cancer Ther. 2015, 14, 2143–2153. [Google Scholar] [CrossRef] [Green Version]
- Vleeshouwer-Neumann, T.; Phelps, M.; Bammler, T.K.; MacDonald, J.W.; Jenkins, I.; Chen, E.Y. Histone Deacetylase Inhibitors Antagonize Distinct Pathways to Suppress Tumorigenesis of Embryonal Rhabdomyosarcoma. PLoS ONE 2015, 10, e0144320. [Google Scholar] [CrossRef] [Green Version]
- Megiorni, F.; Camero, S.; Ceccarelli, S.; McDowell, H.P.; Mannarino, O.; Marampon, F.; Pizer, B.; Shukla, R.; Pizzuti, A.; Marchese, C.; et al. DNMT3B in vitro knocking-down is able to reverse embryonal rhabdomyosarcoma cell phenotype through inhibition of proliferation and induction of myogenic differentiation. Oncotarget 2016, 7, 79342–79356. [Google Scholar] [CrossRef] [Green Version]
- Haydn, T.; Metzger, E.; Schuele, R.; Fulda, S. Concomitant epigenetic targeting of LSD1 and HDAC synergistically induces mitochondrial apoptosis in rhabdomyosarcoma cells. Cell Death Dis. 2017, 8, e2879. [Google Scholar] [CrossRef] [PubMed]
- Filippakopoulos, P.; Picaud, S.; Mangos, M.; Keates, T.; Lambert, J.-P.; Barsyte-Lovejoy, D.; Felletar, I.; Volkmer, R.; Müller, S.; Pawson, T.; et al. Histone Recognition and Large-Scale Structural Analysis of the Human Bromodomain Family. Cell 2012, 149, 214–231. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Cochran, A.G.; Conery, A.R.; Sims, R.J. Bromodomains: A new target class for drug development. Nat. Rev. Drug Discov. 2019, 18, 609–628. [Google Scholar] [CrossRef]
- Anand, P.; Brown, J.D.; Lin, C.Y.; Qi, J.; Zhang, R.; Artero, P.C.; Alaiti, M.A.; Bullard, J.; Alazem, K.; Margulies, K.B.; et al. BET Bromodomains Mediate Transcriptional Pause Release in Heart Failure. Cell 2013, 154, 569–582. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Dey, A.; Chitsaz, F.; Abbasi, A.; Misteli, T.; Ozato, K. The double bromodomain protein Brd4 binds to acetylated chromatin during interphase and mitosis. Proc. Natl. Acad. Sci. USA 2003, 100, 8758–8763. [Google Scholar] [CrossRef] [Green Version]
- Chapuy, B.; McKeown, M.R.; Lin, C.Y.; Monti, S.; Roemer, M.G.M.; Qi, J.; Rahl, P.B.; Sun, H.H.; Yeda, K.T.; Doench, J.G.; et al. Discovery and Characterization of Super-Enhancer-Associated Dependencies in Diffuse Large B Cell Lymphoma. Cancer Cell 2013, 24, 777–790. [Google Scholar] [CrossRef] [Green Version]
- Whitfield, J.R.; Beaulieu, M.E.; Soucek, L. Strategies to Inhibit Myc and Their Clinical Applicability. Front. Cell Dev. Biol. 2017, 5, 10. [Google Scholar] [CrossRef] [Green Version]
- Carabet, L.A.; Rennie, P.S.; Cherkasov, A. Therapeutic Inhibition of Myc in Cancer. Structural Bases and Computer-Aided Drug Discovery Approaches. Int. J. Mol. Sci. 2018, 20, 120. [Google Scholar] [CrossRef] [Green Version]
- Fong, C.Y.; Gilan, O.; Lam, E.Y.N.; Rubin, A.F.; Ftouni, S.; Tyler, D.; Stanley, K.; Sinha, D.; Yeh, P.; Morison, J.; et al. BET inhibitor resistance emerges from leukaemia stem cells. Nature 2015, 525, 538–542. [Google Scholar] [CrossRef]
- Rathert, P.; Roth, M.; Neumann, T.; Muerdter, F.; Roe, J.-S.; Muhar, M.; Deswal, S.; Cerny-Reiterer, S.; Peter, B.; Jude, J.; et al. Transcriptional plasticity promotes primary and acquired resistance to BET inhibition. Nature 2015, 525, 543–547. [Google Scholar] [CrossRef]
- Kurimchak, A.M.; Shelton, C.; Duncan, K.E.; Johnson, K.J.; Brown, J.; O’Brien, S. Resistance to BET Bromodomain Inhibitors Is Mediated by Kinome Reprogramming in Ovarian Cancer. Cell Rep. 2016, 16, 1273–1286. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Shi, X.; Mihaylova, V.T.; Kuruvilla, L.; Chen, F.; Viviano, S.; Baldassarre, M. Loss of TRIM33 causes resistance to BET bromodomain inhibitors through MYC- and TGF-β–dependent mechanisms. Proc. Natl. Acad. Sci. USA 2016, 113, E4558–E4566. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Ma, Y.; Wang, L.; Neitzel, L.R.; Loganathan, S.N.; Tang, N.; Qin, L.; Crispi, E.E.; Guo, Y.; Knapp, S.; Beauchamp, R.D.; et al. The MAPK Pathway Regulates Intrinsic Resistance to BET Inhibitors in Colorectal Cancer. Clin. Cancer Res. 2017, 23, 2027–2037. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Yin, Y.; Sun, M.; Zhan, X.; Wu, C.; Geng, P.; Sun, X.; Wu, Y.; Zhang, S.; Qin, J.; Zhuang, Z.; et al. EGFR signaling confers resistance to BET inhibition in hepatocellular carcinoma through stabilizing oncogenic MYC. J. Exp. Clin. Cancer Res. 2019, 38, 83. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Jang, J.E.; Eom, J.-I.; Jeung, H.-K.; Cheong, J.-W.; Lee, J.Y.; Kim, J.S.; Wu, Y.; Zhang, S.; Qin, J.; Zhuang, Z.; et al. AMPK-ULK1-Mediated Autophagy Confers Resistance to BET Inhibitor JQ1 in Acute Myeloid Leukemia Stem Cells. Clin. Cancer Res. 2017, 23, 2781–2794. [Google Scholar] [CrossRef] [Green Version]
- Lasorsa, E.; Smonksey, M.; Kirk, J.S.; Rosario, S.; Hernandez-Ilizaliturri, F.J.; Ellis, L. Mitochondrial protection impairs BET bromodomain inhibitor-mediated cell death and provides rationale for combination therapeutic strategies. Cell Death Dis. 2015, 6, e2014. [Google Scholar] [CrossRef] [Green Version]
- Lukinavičius, G.; Blaukopf, C.; Pershagen, E.; Schena, A.; Reymond, L.; Derivery, E.; Gonzalez-Gaitan, M.; D’Este, E.; Hell, S.W.; Wolfram Gerlich, D.; et al. SiR-Hoechst is a far-red DNA stain for live-cell nanoscopy. Nat. Commun. 2015, 6, 8497. [Google Scholar] [CrossRef] [Green Version]
- Boyd, M.R.; Paull, K.D. Some practical considerations and applications of the national cancer institute in vitro anticancer drug discovery screen. Drug Dev. Res. 1995, 34, 91–109. [Google Scholar] [CrossRef]
- Vinci, M.; Gowan, S.; Boxall, F.; Patterson, L.; Zimmermann, M.; Court, W.; Lomas, C.; Mendiola, M.; Hardisson, D.; Eccles, S.A. Advances in establishment and analysis of three- dimensional tumor spheroid-based functional assays for target validation and drug evaluation. BMC Biol. 2012, 10, 29. [Google Scholar] [CrossRef] [Green Version]
- Nieddu, V.; Pinna, G.; Marchesi, I.; Sanna, L.; Asproni, B.; Pinna, G.A.; Bagella, L.; Murineddu, G. Synthesis and Antineoplastic Evaluation of Novel Unsymmetrical 1,3,4-Oxadiazoles. J. Med. Chem. 2016, 59, 10451–10469. [Google Scholar] [CrossRef]
- Barretina, J.; Caponigro, G.; Stransky, N.; Venkatesan, K.; Margolin, A.A.; Kim, S.; Wilson, C.J.; Lehár, J.; Kryukov, G.V.; Sonkin, D.; et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 2012, 483, 603–607. [Google Scholar] [CrossRef] [PubMed]
Cell Line | Hours of Treatment | ||
---|---|---|---|
24 | 48 | 72 | |
RD | 3486.5 (2200.4–6405.9) | 431.7 (361.1–511.5) | 103.7 (77.2–134.8) |
A204 | 23.1 (31.5–111.1) | 93.0 (73.6–113.9) | 88.4 (45.6–102.3) |
RH4 | 27.5 (24.9–30.4) | 20.2 (17.5–23.1) | 11.7 (<10–14.2) |
SJCRH30 | 137.8 (52.2–303.7) | 139.4 (102.8–191.5) | 198.2 (146.7–256) |
Cell Line | JQ1 Concentration (nM) | PARP | CLEAVED PARP | MYC | P27 | P21 |
---|---|---|---|---|---|---|
RD | 0 | 1 | 1 | 1 | 1 | 1 |
0.5 | 1.08 | 1.48 | 0.31 | 0.60 | 1.03 | |
1 | 0.62 | 0.77 | 0.19 | 0.44 | 0.90 | |
2.5 | 0.86 | 1.73 | 0.23 | 0.64 | 1.59 | |
A204 | 0 | 1 | 0 | 1 | 1 | 1 |
0.5 | 2.08 | 0.00 | 2.13 | 2.59 | 1.45 | |
1 | 0.89 | 0.00 | 0.93 | 2.34 | 1.19 | |
2.5 | 1.23 | 0.00 | 1.41 | 2.03 | 1.31 | |
RH4 | 0 | 1 | 1 | 1 | 1 | 1 |
0.5 | 0.81 | 0.72 | 0.93 | 1.05 | 1.97 | |
1 | 1.17 | 0.96 | 0.32 | 1.14 | 2.52 | |
2.5 | 1.77 | 3.02 | 0.13 | 1.72 | 1.93 | |
SJCRH30 | 0 | 1 | 1 | 1 | 1 | 1 |
0.5 | 0.74 | 1.52 | 0.71 | 0.87 | 1.84 | |
1 | 0.63 | 4.22 | 0.56 | 0.85 | 2.94 | |
2.5 | 0.61 | 3.87 | 0.32 | 1.06 | 3.76 |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Marchesi, I.; Fais, M.; Fiorentino, F.P.; Bordoni, V.; Sanna, L.; Zoroddu, S.; Bagella, L. Bromodomain Inhibitor JQ1 Provides Novel Insights and Perspectives in Rhabdomyosarcoma Treatment. Int. J. Mol. Sci. 2022, 23, 3581. https://doi.org/10.3390/ijms23073581
Marchesi I, Fais M, Fiorentino FP, Bordoni V, Sanna L, Zoroddu S, Bagella L. Bromodomain Inhibitor JQ1 Provides Novel Insights and Perspectives in Rhabdomyosarcoma Treatment. International Journal of Molecular Sciences. 2022; 23(7):3581. https://doi.org/10.3390/ijms23073581
Chicago/Turabian StyleMarchesi, Irene, Milena Fais, Francesco Paolo Fiorentino, Valentina Bordoni, Luca Sanna, Stefano Zoroddu, and Luigi Bagella. 2022. "Bromodomain Inhibitor JQ1 Provides Novel Insights and Perspectives in Rhabdomyosarcoma Treatment" International Journal of Molecular Sciences 23, no. 7: 3581. https://doi.org/10.3390/ijms23073581