Original ArticlesPiribedil disrupts the MLL1-WDR5 interaction and sensitizes MLL-rearranged acute myeloid leukemia (AML) to doxorubicin-induced apoptosis
Introduction
Histones are the basic units responsible for packing DNA into nucleosomes and are considered carriers of epigenetic information [1]. Post-translational modifications of histone proteins, such as methylation, acetylation, and phosphorylation, are used by multicellular organisms to guarantee the appropriate spatial and temporal expression of key genes during development and differentiation [2,3]. Mixed Lineage Leukemia protein-1 (MLL1) is a histone H3 lysine 4 (H3K4) methyltransferase (HMT) that catalyzes mono-, di-, and tri-methylation through its evolutionarily conserved SET domain and is required for epigenetic maintenance during definitive hematopoiesis due to its regulation of the transcription activation of HOX genes (e.g., HOXA9 and HOXC8), which encode transcription/regulatory factors that promote hematopoietic stem cell expansion [4,5]. A deregulation of MLL1 is often found in both acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML) [6].
According to clinical studies, 5–10% of AML cases in adults and 70% of ALL cases in infants are due to an MLL1 abnormality [7]. In most cases, MLL1 abnormalities involve balanced chromosomal translocations [8]. These translocations produce more than 70 in-frame oncogenic fusion proteins. Over 90% of the transactivation domains of the MLL1 fusion proteins are from AF9, ENL, ELL, AF10, AF4 and AF6 [9]. MLL-r leukemia cells commonly retain and express the other intact allele, i.e., wild-type (WT) MLL1. Despite the loss of the C-terminal SET domain in the MLL1 fusion proteins (MLL-FPs), endogenous MLL1 maintains the global H3K4me status and facilitates MLL-FP-mediated leukemogenesis [10]. According to recent studies, in MLL-r leukemia cells, the oncogenic MLL1-AF9 fusion proteins only target the H3K4me-modified Hox gene locus [11]. The genetic deletion of MLL1 or inhibition of MLL1 methyltransferase activity could induce apoptosis and differentiation in MLL-r leukemia cells [12]. Thus, targeting the H3K4 HMT activity of MLL1 may represent a promising new strategy for the treatment of leukemia patients carrying MLL-FPs.
Because the MLL1 protein alone has extremely low HMT activity, its H3K4 HMT activity is markedly enhanced by assembly into a core complex consisting of the following three other proteins: WDR5, RbBP5 and ASH2L [13]. The interaction between the WDR5 and MLL1 proteins, which is dispensable for the other MLL family proteins, is critical for the structural integrity of the MLL1 core complex and methyltransferase activity [14,15]. Furthermore, the interaction domain structure of these two proteins has been previously defined [16].
Blocking this interaction using small-molecule inhibitors leads to a nearly complete loss of MLL1 methyltransferase activity in vitro but has nearly no effect on the other MLL family HMTs [17]. The first inhibitor of MLL1 enzymatic activities, which was named MM-102, was a peptidomimetic developed in 2012 to target the MLL1-WDR5 interaction [18]. Subsequently, targeting the MLL1 core complex has become increasingly popular in this area of drug discovery [19]. Unfortunately, these inhibitors are all based on the linear peptidomimetic MM-101 as reported previously [20]. This structure-based optimization greatly limits the discovery of new drugs. Moreover, many years of additional investigation are required before these drugs can be applied in the clinic.
To reduce the time-to-market and associated costs and risks, academic researchers aim to identify new targets for old drugs [21]. Piribedil, which is a dopamine D2/D3 agonist, is used for the treatment of patients with Parkinson's disease and circulatory disorders [22]. However, Piribedil has been recently shown to inhibit the growth of colorectal cancer DLD1 cells, suggesting that Piribedil likely has good anti-tumor activity [23]. In this study, we found that Piribedil exhibited a promising anti-leukemic effect on cells harboring MLL-FPs. Specifically, Piribedil disturbs the MLL1-WDR5 interaction and induces changes in gene expression similar to those observed following MLL1 depletion, thereby sensitizing MLL-AML to doxorubicin-based chemotherapy.
Section snippets
Cell culture
The K562 and THP-1 cell lines were cultured in RPMI 1640 (Invitrogen) supplemented with 10% selected FBS (Gibco) and 2 mM l-glutamine. The MV4;11 cell line was maintained in IMDM (Iscove's Modified Dulbecco's Medium) supplemented with 10% selected FBS (Gibco).
Quantitative real-time PCR experiments
The total RNA was isolated from the cells using TRIzol (Invitrogen) and transcribed into cDNA. Real-time PCR was performed on an ABI Prism 7500 Fast Sequence Detection system (Applied Biosystems) using iQTMSYBR1Green Supermix (Bio-Rad).
Piribedil specifically inhibits MLL1 activity and selectively suppresses MLL-r cell proliferation
In order to identify MLL1 inhibitors from a library consists of 592 FDA-approved drugs, we used an in vitro histone methyltransferase assay based on HTRF to screen this library. The scheme was shown in Fig. S1A. As our expected, we found that Piribedil, which has been previously used to treat Parkinson's disease, exerted an extraordinary good activity in inhibiting WT MLL methyltransferase activity (EC50 = 0.18 μM). Notably, the EC50 of Piribedil in the in vitro HTRF assay was close to that of
Discussion
Piribedil has been used for approximately 40 years for the treatment of nervous system disorders, such as Parkinson's disease, intermittent claudication and memory impairment, but has received relatively little attention [30,31]. Drug screening studies have shown that Piribedil exerts multiple therapeutic activities, including anti-cancer activity [32,33]. However, the anti-tumor activity of Piribedil has not been systematically studied. In this study, we found that Piribedil inhibits the
Acknowledgments
Conception and design: Xiong Zhang, Xingling Zheng, Xun Huang; Development of methodology: Xiong Zhang, Xingling Zheng; Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Xiong Zhang; Writing, review, and/or revision of the manuscript: Xiong Zhang, Xun Huang, Mei-yu Geng, Jian Ding; Study supervision: Xun Huang, Mei-yu Geng, Jian Ding.
This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (No.
Conflicts of interest
The authors have no conflicts of interest to declare.
References (45)
- et al.
Multiple interactions recruit MLL1 and MLL1 fusion proteins to the HOXA9 locus in leukemogenesis
Mol. Cell
(2010) - et al.
Pro isomerization in MLL1 PHD3-bromo cassette connects H3K4me readout to CyP33 and HDAC-mediated repression
Cell
(2010) - et al.
Revisiting the biology of infant t(4;11)/MLL-AF4+ B-cell acute lymphoblastic leukemia
Blood
(2015) - et al.
Targeting MLL1 H3K4 methyltransferase activity in mixed-lineage leukemia
Mol. Cell
(2014) - et al.
Structure-based design and synthesis of small molecular inhibitors disturbing the interaction of MLL1-WDR5
Eur. J. Med. Chem.
(2016) - et al.
Controlled inhibition of methyltransferases using photoswitchable peptidomimetics: towards an epigenetic regulation of leukemia
Chem. Sci.
(2017) - et al.
Targeting MLL1 H3K4 methyltransferase activity in mixed-lineage leukemia
Mol. Cell
(2014) - et al.
PRMT4 blocks myeloid differentiation by assembling a methyl-RUNX1-dependent repressor complex
Cell Rep.
(2013) - et al.
Histone acetyltransferase p300/CREB-binding protein-associated factor (PCAF) is required for all-trans-retinoic acid-induced granulocytic differentiation in leukemia cells
J. Biol. Chem.
(2017) - et al.
Genetic abnormalities in myelodysplasia and secondary acute myeloid leukemia: impact on outcome of stem cell transplantation
Blood
(2017)
The H3K4-Methyl epigenome regulates leukemia stem cell oncogenic potential
Canc. Cell
NUP98 fusion proteins interact with the NSL and MLL1 complexes to drive leukemogenesis
Canc. Cell
Cancer epigenetics takes center stage
Proc. Natl. Acad. Sci. U.S.A.
The histone code reader Spin1 controls skeletal muscle development
Cell Death Dis.
Global chromatin profiling reveals NSD2 mutations in pediatric acute lymphoblastic leukemia
Nat. Genet.
The PAF complex regulation of Prmt5 facilitates the progression and maintenance of MLL fusion leukemia
Oncogene
ALOX5 exhibits anti-tumor and drug-sensitizing effects in MLL-rearranged leukemia
Sci. Rep.
Targeted next-gen sequencing for detecting MLL gene fusions in leukemia
Mol. Canc. Res.
MLL1 and MLL1 fusion proteins have distinct functions in regulating leukemic transcription program
Cell discovery
Complementary activities of DOT1L and Menin inhibitors in MLL-rearranged leukemia
Leukemia
Mixed lineage leukemia: a structure-function perspective of the MLL1 protein
FEBS J.
Targeting human SET1/MLL family of proteins
Protein Sci. Publ. Protein Soc.
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These authors contributed equally to this work.