Elucidating the Mechanism of Action of Domatinostat (4SC-202) in Cutaneous T Cell Lymphoma Cells

Overview

Journal J Hematol Oncol

Publisher Biomed Central

Specialties Hematology
Oncology

Date 2019 Mar 20

PMID 30885250

Citations 19

Authors

Marion Wobser

Alexandra Weber

Amelie Glunz

Saskia Tauch

Kristina Seitz

Tobias Butelmann

Sonja Hesbacher

Matthias Goebeler

Rene Bartz

Hella Kohlhof

David Schrama

Roland Houben

Affiliations

Soon will be listed here.

Abstract

Background: Targeting epigenetic modifiers is effective in cutaneous T cell lymphoma (CTCL). However, there is a need for further improvement of this therapeutic approach. Here, we compared the mode of action of romidepsin (FK228), an established class I histone deacetylase inhibitor, and domatinostat (4SC-202), a novel inhibitor of class I HDACs, which has been reported to also target the lysine-specific histone demethylase 1A (LSD1).

Methods: We performed MTS assays and flow cytometric analyses of propidium iodide or annexin V-stained cells to assess drug impact on cellular proliferation, cell cycle distribution, and survival. Histone acetylation and methylation as well as caspase activation was analyzed by immunoblot. Gene expression analysis was performed using NanosString technology. Knockdown and knockout of LSD1 was achieved with shRNA and CRISPR/Cas9, respectively, while the CRISPR/Cas9 synergistic activation mediator system was used to induce expression of endogenous HDACs and LSD1. Furthermore, time-lapse fluorescence microscopy and an in vitro tubulin polymerization assay were applied.

Results: While FK228 as well as 4SC-202 potently induced cell death in six different CTCL cell lines, only in the case of 4SC-202 death was preceded by an accumulation of cells in the G2/M phase of the cell cycle. Surprisingly, apoptosis and accumulation of cells with double DNA content occurred already at 4SC-202 concentrations hardly affecting histone acetylation and methylation, and provoking significantly less changes in gene expression compared to biologically equivalent doses of FK228. Indeed, we provide evidence that the 4SC-202-induced G2/M arrest in CTCL cells is independent of de novo transcription. Furthermore, neither enforced expression of HDAC1 nor knockdown or knockout of LSD1 affected the 4SC-202-induced effects. Since time-lapse microscopy revealed that 4SC-202 could affect mitotic spindle formation, we performed an in vitro tubulin polymerization assay revealing that 4SC-202 can directly inhibit microtubule formation.

Conclusions: We demonstrate that 4SC-202, a drug currently tested in clinical trials, effectively inhibits growth of CTCL cells. The anti-cancer cell activity of 4SC-202 is however not limited to LSD1-inhibition, modulation of histone modifications, and consecutive alteration of gene expression. Indeed, the compound is also a potent microtubule-destabilizing agent.

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References

Rasheed W, Johnstone R, Prince H . Histone deacetylase inhibitors in cancer therapy. Expert Opin Investig Drugs. 2007; 16(5):659-78. DOI: 10.1517/13543784.16.5.659. View

Krejsgaard T, Vetter-Kauczok C, Woetmann A, Lovato P, Labuda T, Eriksen K . Jak3- and JNK-dependent vascular endothelial growth factor expression in cutaneous T-cell lymphoma. Leukemia. 2006; 20(10):1759-66. DOI: 10.1038/sj.leu.2404350. View

Furumai R, Matsuyama A, Kobashi N, Lee K, Nishiyama M, Nakajima H . FK228 (depsipeptide) as a natural prodrug that inhibits class I histone deacetylases. Cancer Res. 2002; 62(17):4916-21. View

McGirt L, Jia P, Baerenwald D, Duszynski R, Dahlman K, Zic J . Whole-genome sequencing reveals oncogenic mutations in mycosis fungoides. Blood. 2015; 126(4):508-19. PMC: 4513251. DOI: 10.1182/blood-2014-11-611194. View

Sheng W, LaFleur M, Nguyen T, Chen S, Chakravarthy A, Conway J . LSD1 Ablation Stimulates Anti-tumor Immunity and Enables Checkpoint Blockade. Cell. 2018; 174(3):549-563.e19. PMC: 6063761. DOI: 10.1016/j.cell.2018.05.052. View

Zhijun H, Shusheng W, Han M, Jianping L, Li-Sen Q, Dechun L . Pre-clinical characterization of 4SC-202, a novel class I HDAC inhibitor, against colorectal cancer cells. Tumour Biol. 2016; 37(8):10257-67. DOI: 10.1007/s13277-016-4868-6. View

Ueda R, Suzuki T, Mino K, Tsumoto H, Nakagawa H, Hasegawa M . Identification of cell-active lysine specific demethylase 1-selective inhibitors. J Am Chem Soc. 2009; 131(48):17536-7. DOI: 10.1021/ja907055q. View

Kalin J, Wu M, Gomez A, Song Y, Das J, Hayward D . Targeting the CoREST complex with dual histone deacetylase and demethylase inhibitors. Nat Commun. 2018; 9(1):53. PMC: 5754352. DOI: 10.1038/s41467-017-02242-4. View

Morrison K, Hergenrother P . Whole cell microtubule analysis by flow cytometry. Anal Biochem. 2011; 420(1):26-32. DOI: 10.1016/j.ab.2011.08.020. View

10.

Angermeyer S, Hesbacher S, Becker J, Schrama D, Houben R . Merkel cell polyomavirus-positive Merkel cell carcinoma cells do not require expression of the viral small T antigen. J Invest Dermatol. 2013; 133(8):2059-64. DOI: 10.1038/jid.2013.82. View

11.

Houben R, Adam C, Baeurle A, Hesbacher S, Grimm J, Angermeyer S . An intact retinoblastoma protein-binding site in Merkel cell polyomavirus large T antigen is required for promoting growth of Merkel cell carcinoma cells. Int J Cancer. 2011; 130(4):847-56. DOI: 10.1002/ijc.26076. View

12.

Lamaa D, Lin H, Zig L, Bauvais C, Bollot G, Bignon J . Design and Synthesis of Tubulin and Histone Deacetylase Inhibitor Based on iso-Combretastatin A-4. J Med Chem. 2018; 61(15):6574-6591. DOI: 10.1021/acs.jmedchem.8b00050. View

13.

Chung C, Poligone B . Cutaneous T cell Lymphoma: an Update on Pathogenesis and Systemic Therapy. Curr Hematol Malig Rep. 2015; 10(4):468-76. DOI: 10.1007/s11899-015-0293-y. View

14.

. StatBite: FDA oncology drug product approvals in 2009. J Natl Cancer Inst. 2010; 102(4):219. DOI: 10.1093/jnci/djq030. View

15.

Weisenberg R . Microtubule formation in vitro in solutions containing low calcium concentrations. Science. 1972; 177(4054):1104-5. DOI: 10.1126/science.177.4054.1104. View

16.

Conte M, De Palma R, Altucci L . HDAC inhibitors as epigenetic regulators for cancer immunotherapy. Int J Biochem Cell Biol. 2018; 98:65-74. DOI: 10.1016/j.biocel.2018.03.004. View

17.

Konermann S, Brigham M, Trevino A, Joung J, Abudayyeh O, Barcena C . Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature. 2014; 517(7536):583-8. PMC: 4420636. DOI: 10.1038/nature14136. View

18.

Huang J, Sengupta R, Espejo A, Lee M, Dorsey J, Richter M . p53 is regulated by the lysine demethylase LSD1. Nature. 2007; 449(7158):105-8. DOI: 10.1038/nature06092. View

19.

Gazdar A, Carney D, Bunn P, RUSSELL E, Jaffe E, Schechter G . Mitogen requirements for the in vitro propagation of cutaneous T-cell lymphomas. Blood. 1980; 55(3):409-17. View

20.

Takagi S, Ishikawa Y, Mizutani A, Iwasaki S, Matsumoto S, Kamada Y . LSD1 Inhibitor T-3775440 Inhibits SCLC Cell Proliferation by Disrupting LSD1 Interactions with SNAG Domain Proteins INSM1 and GFI1B. Cancer Res. 2017; 77(17):4652-4662. DOI: 10.1158/0008-5472.CAN-16-3502. View