Small molecule inhibition of lysine-specific demethylase 1 (LSD1) and histone deacetylase (HDAC) alone and in combination in Ewing sarcoma cell lines

Autoři: Darcy Welch aff001;  Elliot Kahen aff001;  Brooke Fridley aff003;  Andrew S. Brohl aff004;  Christopher L. Cubitt aff002;  Damon R. Reed aff001
Působiště autorů: Sunshine Lab, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, United States of America aff001;  Translational Research Core, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, United States of America aff002;  Department of Biostatistics, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, United States of America aff003;  Sarcoma Department, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, United States of America aff004;  Chemical Biology and Molecular Medicine Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, United States of America aff005;  Adolescent and Young Adult Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, United States of America aff006
Vyšlo v časopise: PLoS ONE 14(9)
Kategorie: Research Article
doi: 10.1371/journal.pone.0222228


Ewing Sarcoma (ES) is characterized by recurrent translocations between EWSR1 and members of the ETS family of transcription factors. The transcriptional activity of the fusion oncoprotein is dependent on interaction with the nucleosome remodeling and deactylase (NuRD) co-repressor complex. While inhibitors of both histone deacetylase (HDAC) and lysine-specific demethylase-1 (LSD1) subunits of the NuRD complex demonstrate single agent activity in preclinical models, combination strategies have not been investigated. We selected 7 clinically utilized chemotherapy agents, or active metabolites thereof, for experimentation: doxorubicin, cyclophosphamide, vincristine, etoposide and irinotecan as well as the HDAC inhibitor romidepsin and the reversible LSD1 inhibitor SP2509. All agents were tested at clinically achievable concentrations in 4 ES cell lines. All possible 2 drug combinations were then tested for potential synergy. Order of addition of second-line conventional combination therapy agents was tested with the addition of SP2509. In two drug experiments, synergy was observed with several combinations, including when SP2509 was paired with topoisomerase inhibitors or romidepsin. Addition of SP2509 after treatment with second-line combination therapy agents enhanced treatment effect. Our findings suggest promising combination treatment strategies that utilize epigenetic agents in ES.

Klíčová slova:

Cell viability testing – Cyclophosphamide – Drug metabolism – Drug therapy – Chemotherapeutic agents – Chemotherapy – Ewing sarcoma – Synergy testing


1. Gorlick R, Janeway K, Lessnick S, Randall RL, Marina N, Committee COGBT. Children's Oncology Group's 2013 blueprint for research: bone tumors. Pediatr Blood Cancer. 2013;60(6):1009–15. doi: 10.1002/pbc.24429 23255238; PubMed Central PMCID: PMC4610028.

2. Delattre O, Zucman J, Plougastel B, Desmaze C, Melot T, Peter M, et al. Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours. Nature. 1992;359(6391):162–5. doi: 10.1038/359162a0 1522903

3. Sankar S, Bell R, Stephens B, Zhuo R, Sharma S, Bearss DJ, et al. Mechanism and relevance of EWS/FLI-mediated transcriptional repression in Ewing sarcoma. Oncogene. 2013;32(42):5089–100. doi: 10.1038/onc.2012.525 23178492; PubMed Central PMCID: PMC3899696.

4. Brohl AS, Solomon DA, Chang W, Wang J, Song Y, Sindiri S, et al. The genomic landscape of the Ewing Sarcoma family of tumors reveals recurrent STAG2 mutation. PLoS Genet. 2014;10(7):e1004475. doi: 10.1371/journal.pgen.1004475 25010205; PubMed Central PMCID: PMC4091782.

5. Crompton BD, Stewart C, Taylor-Weiner A, Alexe G, Kurek KC, Calicchio ML, et al. The genomic landscape of pediatric Ewing sarcoma. Cancer Discov. 2014;4(11):1326–41. doi: 10.1158/2159-8290.CD-13-1037 25186949.

6. Tirode F, Surdez D, Ma X, Parker M, Le Deley MC, Bahrami A, et al. Genomic Landscape of Ewing Sarcoma Defines an Aggressive Subtype with Co-Association of STAG2 and TP53 Mutations. Cancer Discovery. 2014;4(11):1342–53. doi: 10.1158/2159-8290.CD-14-0622 25223734

7. Erkizan HV, Kong Y, Merchant M, Schlottmann S, Barber-Rotenberg JS, Yuan L, et al. A small molecule blocking oncogenic protein EWS-FLI1 interaction with RNA helicase A inhibits growth of Ewing's sarcoma. Nat Med. 2009;15(7):750–6. doi: 10.1038/nm.1983 19584866; PubMed Central PMCID: PMC2777681.

8. Wang Y, Zhang H, Chen Y, Sun Y, Yang F, Yu W, et al. LSD1 is a subunit of the NuRD complex and targets the metastasis programs in breast cancer. Cell. 2009;138(4):660–72. Epub 2009/08/26. doi: 10.1016/j.cell.2009.05.050 19703393.

9. Feng Z, Yao Y, Zhou C, Chen F, Wu F, Wei L, et al. Pharmacological inhibition of LSD1 for the treatment of MLL-rearranged leukemia. J Hematol Oncol. 2016;9:24. Epub 2016/03/15. doi: 10.1186/s13045-016-0252-7 26970896; PubMed Central PMCID: PMC4789278.

10. Alsaquer SF, Tashkandi MM, Kartha VK, Yang Y-T, Alkheriji Y, Salama A, et al. Inhibition of lsd1 epigenetically attenuates oral cancer growth and metastasis. Oncotarget. 2017;8(43):73372–86. Epub 07/27/17. doi: 10.18632/oncotarget.19637 PubMed Central PMCID: PMC5650269. 29088714

11. Fang J, Landersdorfer CB, Cirincione B, Jusko WJ. Study reanalysis using a mechanism-based pharmacokinetic/pharmacodynamic model of pramlintide in subjects with type 1 diabetes. AAPS J. 2013;15(1):15–29. doi: 10.1208/s12248-012-9409-7 23054970; PubMed Central PMCID: PMC3535104.

12. Sankar S, Theisen ER, Bearss J, Mulvihill T, Hoffman LM, Sorna V, et al. Reversible LSD1 inhibition interferes with global EWS/ETS transcriptional activity and impedes Ewing sarcoma tumor growth. Clin Cancer Res. 2014;20(17):4584–97. doi: 10.1158/1078-0432.CCR-14-0072 24963049; PubMed Central PMCID: PMC4155010.

13. McGrath JP, Williamson KE, Balasubramanian S, Odate S, Arora S, Hatton C, et al. Pharmacological Inhibition of the Histone Lysine Demethylase KDM1A Suppresses the Growth of Multiple Acute Myeloid Leukemia Subtypes. Cancer Res. 2016;76(7):1975–88. Epub 2016/02/04. doi: 10.1158/0008-5472.CAN-15-2333 26837761.

14. Sehrawat A, Gao L, Wang Y, Bankhead A, 3rd, McWeeney SK, King CJ, et al. LSD1 activates a lethal prostate cancer gene network independently of its demethylase function. Proc Natl Acad Sci U S A. 2018;115(18):E4179–E88. Epub 2018/03/28. doi: 10.1073/pnas.1719168115 29581250; PubMed Central PMCID: PMC5939079.

15. Mohammad HP, Smitheman KN, Kamat CD, Soong D, Federowicz KE, Van Aller GS, et al. A DNA Hypomethylation Signature Predicts Antitumor Activity of LSD1 Inhibitors in SCLC. Cancer Cell. 2015;28(1):57–69. Epub 2015/07/16. doi: 10.1016/j.ccell.2015.06.002 26175415.

16. Maes T, Mascaro C, Tirapu I, Estiarte A, Ciceri F, Lunardi S, et al. ORY-1001, a Potent and Selective Covalent KDM1A Inhibitor, for the Treatment of Acute Leukemia. Cancer Cell. 2018;33(3):495–511 e12. Epub 2018/03/06. doi: 10.1016/j.ccell.2018.02.002 29502954.

17. Romo-Morales A, Aladowicz E, Blagg J, Gatz SA, Shipley JM. Catalytic inhibition of KDM1A in Ewing sarcoma is insufficient as a therapeutic strategy. Pediatr Blood Cancer. 2019;66(9):e27888. Epub 2019/06/18. doi: 10.1002/pbc.27888 31207107.

18. Sonnemann J, Zimmerman M, Marx C, Ebert F, Becker S, Lauterjung M-L, et al. LSDI (KMDIA)-independent effects of the LSD1 inhibitor SP2509 in cancer cells. British Journal of Haemetology. 2018;183:491–524. Epub 12/03/17. doi: 10.1111/bjh.14983 PubMed Central PMCID: PMC 29205263 29205263

19. Sonnemann J, Dreyer L, Hartwig M, Palani CD, Hong LTT, Klier U, et al. Histone deacetylase inhibitors induce cell death and enhance the apoptosis-inducing activity of TRAIL in Ewing’s sarcoma cells. Journal of Cancer Research and Clinical Oncology. 2007;133(11):847–58. doi: 10.1007/s00432-007-0227-8 17486365

20. Shi YJ, Matson C, Lan F, Iwase S, Baba T, Shi Y. Regulation of LSD1 histone demethylase activity by its associated factors. Mol Cell. 2005;19(6):857–64. Epub 2005/09/06. doi: 10.1016/j.molcel.2005.08.027 16140033.

21. Thomas S, Aggarwal R, Jahan T, Ryan C, Troung T, Cripps AM, et al. A phase I trial of panobinostat and epirubicin in solid tumors with a dose expansion in patients with sarcoma. Ann Oncol. 2016;27(5):947–52. Epub 2016/02/24. doi: 10.1093/annonc/mdw044 26903311; PubMed Central PMCID: PMC4843187.

22. Cassier PA, Lefranc A, Amela EY, Chevreau C, Bui BN, Lecesne A, et al. A phase II trial of panobinostat in patients with advanced pretreated soft tissue sarcoma. A study from the French Sarcoma Group. Br J Cancer. 2013;109(4):909–14. Epub 2013/08/08. doi: 10.1038/bjc.2013.442 23922114; PubMed Central PMCID: PMC3749588.

23. Kahen E, Yu D, Harrison DJ, Clark J, Hingorani P, Cubitt CL, et al. Identification of clinically achievable combination therapies in childhood rhabdomyosarcoma. Cancer Chemother Pharmacol. 2016;78(2):313–23. Epub 2016/06/22. doi: 10.1007/s00280-016-3077-8 27324022; PubMed Central PMCID: PMC4965487.

24. Yu D, Kahen E, Cubitt CL, McGuire J, Kreahling J, Lee J, et al. Identification of Synergistic, Clinically Achievable, Combination Therapies for Osteosarcoma. Sci Rep. 2015;5:16991. Epub 2015/11/26. doi: 10.1038/srep16991 26601688; PubMed Central PMCID: PMC4658502.

25. Goldsby RE, Fan TM, Villaluna D, Wagner LM, Isakoff MS, Meyer J, et al. Feasibility and dose discovery analysis of zoledronic acid with concurrent chemotherapy in the treatment of newly diagnosed metastatic osteosarcoma: a report from the Children's Oncology Group. Eur J Cancer. 2013;49(10):2384–91. Epub 2013/05/15. doi: 10.1016/j.ejca.2013.03.018 23664013; PubMed Central PMCID: PMC3689577.

26. Meyers PA, Schwartz CL, Krailo M, Kleinerman ES, Betcher D, Bernstein ML, et al. Osteosarcoma: a randomized, prospective trial of the addition of ifosfamide and/or muramyl tripeptide to cisplatin, doxorubicin, and high-dose methotrexate. J Clin Oncol. 2005;23(9):2004–11. Epub 2005/03/19. doi: 10.1200/JCO.2005.06.031 15774791.

27. Pishas KI, Drenberg CD, Taslim C, Theisen ER, Johnson KM, Saund RS, et al. Therapeutic Targeting of KDM1A/LSD1 in Ewing Sarcoma with SP-2509 Engages the Endoplasmic Reticulum Stress Response. Molecular Cancer Therapeutics. 2018;17(9):1902–16. doi: 10.1158/1535-7163.MCT-18-0373 29997151

28. El-Najjar N, Ketola RA, Nissila T, Mauriala T, Antopolsky M, Janis J, et al. Impact of protein binding on the analytical detectability and anticancer activity of thymoquinone. J Chem Biol. 2011;4(3):97–107. Epub 2012/01/10. doi: 10.1007/s12154-010-0052-4 22229047; PubMed Central PMCID: PMC3124627.

29. Chou T-C. Theoretical Basis, Experimental Design, and Computerized Simulation of Synergism and Antagonism in Drug Combination Studies. Pharmacological Reviews. 2006;58(3):621–81. doi: 10.1124/pr.58.3.10 16968952

30. Wagner LM. Fifteen years of irinotecan therapy for pediatric sarcoma: where to next? Clin Sarcoma Res. 2015;5:20. Epub 2015/09/01. doi: 10.1186/s13569-015-0035-x 26322224; PubMed Central PMCID: PMC4552408.

31. Palmerini E, Jones RL, Setola E, Picci P, Marchesi E, Luksch R, et al. Irinotecan and temozolomide in recurrent Ewing sarcoma: an analysis in 51 adult and pediatric patients. Acta Oncol. 2018;57(7):958–64. Epub 2018/03/14. doi: 10.1080/0284186X.2018.1449250 29533113.

32. Crompton BD, Stewart C, Taylor-Weiner A, Alexe G, Kurek KC, Calicchio ML, et al. The Genomic Landscape of Pediatric Ewing Sarcoma. Cancer Discovery. 2014;4(11):1326–41. doi: 10.1158/2159-8290.CD-13-1037 25186949

33. Womer RB, West DC, Krailo MD, Dickman PS, Pawel BR, Grier HE, et al. Randomized controlled trial of interval-compressed chemotherapy for the treatment of localized Ewing sarcoma: a report from the Children's Oncology Group. J Clin Oncol. 2012;30(33):4148–54. doi: 10.1200/JCO.2011.41.5703 23091096; PubMed Central PMCID: PMC3494838.

34. Reed DR, Hayashi M, Wagner L, Binitie O, Steppan DA, Brohl AS, et al. Treatment pathway of bone sarcoma in children, adolescents, and young adults. Cancer. 2017;123(12):2206–18. doi: 10.1002/cncr.30589 28323337; PubMed Central PMCID: PMC5485018.

35. Rudolph T, Yonezawa M, Lein S, Heidrich K, Kubicek S, Schafer C, et al. Heterochromatin formation in Drosophila is initiated through active removal of H3K4 methylation by the LSD1 homolog SU(VAR)3-3. Mol Cell. 2007;26(1):103–15. Epub 2007/04/17. doi: 10.1016/j.molcel.2007.02.025 17434130.

36. Hayami S, Kelly JD, Cho HS, Yoshimatsu M, Unoki M, Tsunoda T, et al. Overexpression of LSD1 contributes to human carcinogenesis through chromatin regulation in various cancers. Int J Cancer. 2011;128(3):574–86. Epub 2010/03/25. doi: 10.1002/ijc.25349 20333681.

37. Zöllner SK, Selvanathan SP, Graham GT, Commins RMT, Hong SH, Moseley E, et al. Inhibition of the oncogenic fusion protein EWS-FLI1 causes G2-M cell cycle arrest and enhanced vincristine sensitivity in Ewing’s sarcoma. Science Signaling. 2017;10(499). doi: 10.1126/scisignal.aam8429 28974650

38. May WA, Grigoryan RS, Keshelava N, Cabral DJ, Christensen LL, Jenabi J, et al. Characterization and drug resistance patterns of Ewing's sarcoma family tumor cell lines. PLoS One. 2013;8(12):e80060. doi: 10.1371/journal.pone.0080060 24312454; PubMed Central PMCID: PMC3846563.

39. Franzetti GA, Laud-Duval K, van der Ent W, Brisac A, Irondelle M, Aubert S, et al. Cell-to-cell heterogeneity of EWSR1-FLI1 activity determines proliferation/migration choices in Ewing sarcoma cells. Oncogene. 2017;36(25):3505–14. Epub 2017/01/31. doi: 10.1038/onc.2016.498 28135250; PubMed Central PMCID: PMC5541267.

40. Gupta S, Weston A, Bearrs J, Thode T, Neiss A, Soldi R, et al. Reversible lysine-specific demethylase 1 antagonist HCI-2509 inhibits growth and decreases c-MYC in castration- and docetaxel-resistant prostate cancer cells. Prostate Cancer Prostatic Dis. 2016;19(4):349–57. Epub 2016/06/29. doi: 10.1038/pcan.2016.21 27349498; PubMed Central PMCID: PMC5133270 holder of Salarius Pharmaceuticals. SG's spouse is an equity holder of Salarius Pharmaceuticals. The remaining authors declare no conflicts of interest.

41. Sato T, Cesaroni M, Chung W, Panjarian S, Tran A, Madzo J, et al. Transcriptional Selectivity of Epigenetic Therapy in Cancer. Cancer Research. 2017;77(2):470–81. doi: 10.1158/0008-5472.CAN-16-0834 27879268

42. 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(6):e2879. Epub 2017/06/16. doi: 10.1038/cddis.2017.239 28617441; PubMed Central PMCID: PMC5520898.

43. Fiskus W, Sharma S, Shah B, Portier BP, Devaraj SG, Liu K, et al. Highly effective combination of LSD1 (KDM1A) antagonist and pan-histone deacetylase inhibitor against human AML cells. Leukemia. 2014;28(11):2155–64. Epub 2014/04/05. doi: 10.1038/leu.2014.119 24699304; PubMed Central PMCID: PMC4739780.

44. Kalin JH, Wu M, Gomez AV, Song Y, Das J, Hayward D, et al. Targeting the CoREST complex with dual histone deacetylase and demethylase inhibitors. Nat Commun. 2018;9(1):53. Epub 2018/01/06. doi: 10.1038/s41467-017-02242-4 29302039; PubMed Central PMCID: PMC5754352.

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