Dot1L Inhibitor EPZ-5676: Synthesis, pharmacokinetic and tissue distribution studies in Sprague-Dawley rats

Main Article Content

Vijay Kumar Michael F Wempe Janet W. Lightner Peter J. Rice Marielle Nebout Jean-Francois Peyron


Histone methyl-transferase Dot1L can methylate histone 3 on lysine 79 (H3K79). Herein we present the chemical synthesis of a cis/trans mixture of a potent Dot1L inhibitor known as EPZ-5676 (2). Upon preparing compound 2, we tested the compound in mixed and non-mixed lineage leukemia cell lines. The two MLL-rearranged cell lines were MV4;11 and Molm14; whereas the two non-MLL-rearranged (control) cell lines were Molt4 and Kasumi. We observed anticipated in vitro activity for compound 2 in these four leukemia cell lines; results illustrating that Dot1L inhibition can trigger cancer cell death.  In addition, we also tested 2 in a new leukemia cell line known as KO99L; KO99L cells have been shown to over express the membrane amino acid transporter known as L-amino acid type 1 (LAT1). Compound 2 was also observed to decrease cell viability in the KO99L cell; albeit at higher concentrations as compared to the MLL-rearranged cell lines. In addition to in vitro experiments, we also performed in vivo experiments in Sprague-Dawley rats.  Intravenous (i.e. orbital sinus dosing) experiments were performed at two different doses (i.e. 1.0 and 2.0 mg/kg). These rat Pharmacokinetic (PK) results indicate that compound 2 has a slow distribution phase, followed by an extended terminal half-life (i.e. 11.2 ± 3.1 h).  Furthermore, tissue distribution experiments demonstrate that 2 predominately distributes to kidney, blood and liver, and to a limited extend, was detectable in brain.

Article Details

How to Cite
KUMAR, Vijay et al. Dot1L Inhibitor EPZ-5676: Synthesis, pharmacokinetic and tissue distribution studies in Sprague-Dawley rats. Medical Research Archives, [S.l.], n. 3, july 2015. ISSN 2375-1924. Available at: <>. Date accessed: 23 may 2024.
Research Articles


Anglin, J. L.; Deng, L.; Yao, Y.; Cai, G.; Liu, Z.; Jiang, H. 2012. Synthesis and structure-activity relationship investigation of adenosine-containing inhibitors of histone methyltransferase DOT1L. J Med Chem., 55:8066-8074.

Basavapathruni, A.; Olhava, E. J.; Daigle, S. R.; Therkelsen, C. A.; Jin, L.; Boriack-Sjodin, P. A. 2014. Nonclinical pharmacokinetics and metabolism of EPZ-5676, a novel DOT1L histone methyltransferase inhibitor. Biopharm Drug Dispos., 35:237-252.

Bernt, K. M.; Zhu, N.; Sinha, A. U.; Vempati, S.; Faber, J.; Krivtsov, A. V. 2011. MLL-rearranged leukemia is dependent on aberrant H3K79 methylation by DOT1L. Cancer Cell., 20:66-78.

Bitoun, E.; Oliver, P. L.; Davies, K. E. 2007. The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling. Hum. Mol. Gen.,16:92-106.

Chang, M. J.; Wu, H.; Achille, N. J.; Reisenauer, M. R.; Chou, C. W.; Zeleznik-Le, N. J. 2010. Histone H3 lysine 79 methyltransferase Dot1 is required for immortalization by MLL oncogenes. Cancer Res., 70:10234-10242.

Chen, L.; Deshpande, A. J.; Banka, D.; Bernt, K. M.; Dias, S.; Buske, C. 2013. Abrogation of MLL-AF10 and CALM-AF10-mediated transformation through genetic inactivation or pharmacological inhibition of the H3K79 methyltransferase Dot1l. Leukemia., 27:813-22.

Chen, C-W.; Kocke, R.P.; Sinha, A.U.; Deshpande, A.J.; Zhu, N.; Eng, R.; Doench, J.G.; Xu, H.; Chu, S.H.; Qi, J.; Wang, X.; Delaney, C.; Bernt, K.M.; Root, D.E.; Hahn, W.C.; Bradner, J.E.; Armstrong, S.A. 2015. DOT1L inhibits SIRT1-mediated epigenetic silencing to maintain leukemic gene expression in MLL-rearranged leukemia. Nature Medicine., 21: 335-343.

Daigle, S. R.; Olhava, E. J.; Therkelsen, C. A.; Majer, C. R.; Sneeringer, C. J.; Song, J. 2011. Selective killing of mixed lineage leukemia cells by a potent small-molecule DOT1L inhibitor. Cancer Cell., 20:53-65.

Daigle, S. R.; Olhava, E. J.; Therkelsen, C. A.; Basavapathruni, A.; Jin, L.; Boriack-Sjodin, P. A. 2013. Potent inhibition of DOT1L as treatment of MLL-fusion leukemia. Blood., 122:1017-1025.

Deshpande, A. J.; Chen, L.; Fazio, M.; Sinha, A. U.; Bernt, K. M.; Banka, D. 2013. Leukemic transformation by the MLL-AF6 fusion oncogene requires the H3K79 methyltransferase Dot1l. Blood., 121:2533-2541.

Dlakic, M. 2001. Chromatin silencing protein and pachytene checkpoint regulator Dot1p has a methyltransferase fold. Trends Biochem Sci.,26:405-407.

Ho, L. L.; Sinha, A.; Verzi, M.; Bernt, K. M.; Armstrong, S. A.; Shivdasani, R. A. 2013. DOT1L-mediated H3K79 methylation in chromatin is dispensable for Wnt pathway-specific and other intestinal epithelial functions. Mol Cell Biol., 33:1735-1745.

Jo, S. Y.; Granowicz, E. M.; Maillard, I.; Thomas, D.; Hess, J. L. 2011. Requirement for Dot1l in murine postnatal hematopoiesis and leukemogenesis by MLL translocation. Blood., 117:4759-4768.

Jones, B.; Su, H.; Bhat, A.; Lei, H.; Bajko, J.; Hevi, S. 2008. The histone H3K79 methyltransferase Dot1L is essential for mammalian development and heterochromatin structure. PLoS Genet., 4:e1000190.

Klaus, C.R.; Iwanowicz, D.; Johnston, D.; Campbell, C.A.; Smith, J.J.; Moyer, M.P.; Copeland, R.A.; Olhava, E.J.; Scott, M.P.; Pollock, R.M.; Daigle, S.R.; Raimondi, A. 2014. DOT1L Inhibitor EPZ-5676 Displays Synergistic Antiproliferative Activity in Combination with Standard of Care Drugs and Hypomethylating Agents in MLL-Rearranged Leukemia Cells. JPET., 350: 646-656.

Mahmoudi, T.; Boj, S. F.; Hatzis, P.; Li, V. S.; Taouatas, N.; Vries, R. G. 2010. The leukemia-associated Mllt10/Af10-Dot1l are Tcf4/beta-catenin coactivators essential for intestinal homeostasis. PLoS Biol., 8:e1000539.
Min, J.; Feng, Q.; Li, Z.; Zhang, Y.; Xu, R. M. 2003.

Structure of the catalytic domain of human DOT1L, a non-SET domain nucleosomal histone methyltransferase. Cell., 112:711-723.

Mueller, D.; Bach, C.; Zeisig, D.; Garcia-Cuellar, M. P.; Monroe, S.; Sreekumar, A. 2007. A role for the MLL fusion partner ENL in transcriptional elongation and chromatin modification. Blood., 110:4445-4454.

Mueller, D.; Garcia-Cuellar, M. P.; Bach, C.; Buhl, S.; Maethner, E.; Slany, R. K. 2009. Misguided transcriptional elongation causes mixed lineage leukemia. PLoS Biol. 7:e1000249.

Ng, H. H.; Feng, Q.; Wang, H.; Erdjument-Bromage, H.; Tempst, P.; Zhang, Y. 2002. Lysine methylation within the globular domain of histone H3 by Dot1 is important for telomeric silencing and Sir protein association. Genes Dev., 16:1518–1527.

Nguyen, A. T.; He, J.; Taranova, O.; Zhang, Y. 2011(a). Essential role of DOT1L in maintaining normal adult hematopoiesis. Cell Res.,21:1370-1373.

Nguyen, A. T.; Taranova, O.; He, J.; Zhang, Y. 2011(b). DOT1L, the H3K79 methyltransferase, is required for MLL-AF9-mediated leukemogenesis. Blood., 117:6912-6922.

Nguyen, A. T.; Xiao, B.; Neppl, R. L.; Kallin, E. M.; Li, J.; Chen, T. 2011(c). DOT1L regulates dystrophin expression and is critical for cardiac function. Genes Dev., 25:263-274.

Nguyen, A. T.; Zhang, Y. 2011(d). The diverse functions of Dot1 and H3K79 methylation. Genes Dev., 25:1345-58. doi: 10.1101/gad.2057811

Okada, Y.; Feng, Q.; Lin, Y.; Jiang, Q.; Li, Y.; Coffield, V. M. 2005. hDOT1L links histone methylation to leukemogenesis. Cell., 121:167-78.

Olhava, J.E.; Chesworth, R.; Kuntz, W.K.; Richon, M.V.; Pollock, M.R. 2012. International Application Published Under The Patent Cooperation Treaty (PCT). WO 2012/075381 A1.

Olhava, J.E.; Chesworth, R.; Kuntz, W.K.; Richon, M.V.; Pollock, M.R.; Daigle, R.S. 2014(a). International Application Published Under The Patent Cooperation Treaty (PCT). WO 2014/039839 A1.

Onder, T. T.; Kara, N.; Cherry, A.; Sinha, A. U.; Zhu, N.; Bernt, K. M. 2012. Chromatin-modifying enzymes as modulators of reprogramming. Nature., 483:598-602.

Park, J. H.; Cosgrove, M. S.; Youngman, E.; Wolberger, C.; Boeke, J. D. 2002. A core nucleosome surface crucial for transcriptional silencing. Nat Genet.,32:273-279.

Rosilio, C.; Nebout, M.; Imbert, V.; Griessinger, E.; Neffati, Z.; Benadiba, J.; Hagenbeek, T.; Spits, H.; Reverso, J.; Ambrosetti, D.; Michiels, J. F.; Bailly-Maitre, B.; Endou, H.; Wempe, M. F.; Peyron, J. F. 2015. Leukemia., in press. Dec 8. doi: 10.1038/leu.2014.338. [Epub ahead of print] PMID: 25482130.

Sawada, K.; Yang, Z.; Horton, J. R.; Collins, R. E.; Zhang, X.; Cheng, X. 2004. Structure of the conserved core of the yeast Dot1p, a nucleosomal histone H3 lysine 79 methyltransferase. J Biol Chem., 279:43296-43306.

Segawa, H.; Fukasawa, Y.; Miyamoto, K.; Takeda, E.; Endou, H.; Kanai, Y. 1999. Identification and functional characterization of a Na+-independent neutral amino acid transporter with broad substrate selectivity. J Biol Chem., 274:19745-19751.

Steger, D. J.; Lefterova, M. I.; Ying, L.; Stonestrom, A. J.; Schupp, M.; Zhuo, D. 2008. DOT1L/KMT4 recruitment and H3K79 methylation are ubiquitously coupled with gene transcription in mammalian cells. Mol Cell Biol., 28:2825-2839.

van Leeuwen, F.; Gafken, P. R.; Gottschling, D. E. 2002. Dot1p modulates silencing in yeast by methylation of the nucleosome core. Cell., 109:745-56.

Wempe, M. F.; Lightner, J. W.; Miller, B.; Iwen, T. J.; Rice, P. J.; Wakui, A.; Anzai, N.; Jutabha, P.; Endou, H. 2012. Potent human uric acid transporter 1 inhibitors: in vitro and in vivo metabolism and pharmacokinetic studies. Drug Design, Development and Therapy, 6:323-339.

Yanagida, O.; Kanai, Y.; Chairoungdua, A.; Kim, D. K.; Segawa, H.; Nii, T.; Cha, S. H.; Matsuo, H.; Fukushima, J.; Fukasawa, Y.; Tani, Y.; Taketani, Y.; Uchino, H.; Kim, J. Y.; Inatomi, J.; Okayasu, I.; Miyamoto, K.; Takeda, E.; Goya, T.; Endou, H. 2001. Human L-type amino acid transporter 1 (LAT1): characterization of function and expression in tumor cell lines. Biochim Biophys Acta., 1514:291-302.

Yang, L.; Lin, C.; Jin, C.; Yang, J. C.; Tanasa, B.; Li, W. 2013. lncRNA-dependent mechanisms of androgen-receptor-regulated gene activation programs. Nature., 500:598-602.

Yu, W.; Chory, E. J.; Wernimont, A. K.; Tempel, W.; Scopton, A.; Federation, A. 2012. Catalytic site remodelling of the DOT1L methyltransferase by selective inhibitors. Nat Commun., 3:1288.

Yu, W.; Smil, D.; Li, F.; Tempel, W.; Fedorov, O.; Nguyen, K. T. 2013. Bromo-deaza-SAH: a potent and selective DOT1L inhibitor. Bioorg Med Chem., 21:1787-1794.

Most read articles by the same author(s)