Cancer Letters

Cancer Letters

Volume 431, 1 September 2018, Pages 150-160
Cancer Letters

Original Articles
Piribedil disrupts the MLL1-WDR5 interaction and sensitizes MLL-rearranged acute myeloid leukemia (AML) to doxorubicin-induced apoptosis

https://doi.org/10.1016/j.canlet.2018.05.034Get rights and content

Highlights

  • Piribedil selectively suppresses MLL-r cell proliferation.

  • Piribedil selectively decreases the H3K4 methylation profile and disturbs the MLL1-WDR5 interaction.

  • Piribedil downregulates the expression of MLL1 target genes and induces cell-cycle arrest, apoptosis and differentiation in MLL-r cells.

  • Piribedil inhibits MLL-r tumor growth and decreases the expression of MLL1 target genes in vivo; and.

  • Piribedil sensitizes MLL-r AML cells to doxorubicin-induced apoptosis.

Abstract

Targeting WT MLL for the treatment of MLL-r leukemia, which is highly aggressive and resistant to chemotherapy, has been shown to be a promising strategy. However, drug treatments targeting WT MLL are lacking. We used an in vitro histone methyltransferase assay to screen a library consists of 592 FDA–approved drugs for MLL1 inhibitors by measuring alterations in HTRF signal and found that Piribedil represented a potent activity. Piribedil specifically inhibited the proliferation of MLL-r cells by inducing cell-cycle arrest, apoptosis and myeloid differentiation with little toxicity to the non-MLL cells. Mechanism study showed Piribedil blocked the MLL1-WDR5 interaction and thus selectively reduced MLL1-dependent H3K4 methylation. Importantly, MLL1 depletion induced gene expression that was similar to that induced by Piribedil and rendered the MLL-r cells resistant to Piribedil-induced toxicity, revealing Piribedil exerted anti-leukemia effects by targeting MLL1. Furthermore, both the Piribedil treatment and MLL1 depletion sensitized the MLL-r cells to doxorubicin-induced apoptosis. Our study support the hypothesis that Piribedil could serve as a new drug for the treatment of MLL-r AML and provide new insight for further optimization of targeting MLL1 HMT activity.

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.

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