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K. Ishiguro et al.
ciated with active transcription, while methylation of H3K9 and H3K27 are well known to be repressive marks.7,8 Moreover, dysregulation of histone methylation appears to be involved in the pathogenesis of MM. Mutations in genes encoding the histone modifiers H3K27 demethylase UTX (also known as KDM6A); H3K4 methyltransferases MLL, MLL2, and MLL3; H3K9 methyl- transferase G9a (also known as EHMT2); and H3K36 methyltransferase MMSET (also known as WHSC1 or NSD2) have been detected in MM.9,10 MMSET is overex- pressed in MM with t(4;14), which leads to a global accu- mulation of H3K36 dimethylation (H3K36me2) and reduction of H3K27me3.11 EZH2 is also reportedly overex- pressed in MM, is associated with a poor prognosis, and is considered a potential therapeutic target.12,13 In the present study, we aimed to examine the pathological and thera- peutic implications of histone methylation in MM.
Methods
Cell lines and clinical specimens
MM cell lines (RPMI-8226, MM.1S, KMS-11, KMS-12BM, KMS-12PE and U-266) were obtained and cultured as described previously.14 All cell lines were authenticated using short tandem repeat analysis performed by JCRB (Tokyo, Japan) or BEX (Tokyo, Japan) between 2015 and 2017. Total RNA and genomic DNA were extracted using RNeasy Mini Kits (Qiagen, Hilden, Germany) and QIAamp DNA Mini Kits (Qiagen) according to the manufacturer’s instructions. Specimens of bone marrow or periph- eral blood were respectively collected from MM or plasma cell leukemia (PCL) patients, after which CD138-positive cells were isolated using a MACS manual cell separator (Miltenyi Biotec, Bergisch Gladbach, Germany). CD138-positive cells were cultured for 24 hours in RPMI-1640 medium supplemented with 20% fetal bovine serum (FBS) and 1% penicillin/streptomycin/amphotericin B, after which drug treatment and cell viability assays were per- formed. This study was performed in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of Sapporo Medical University. Informed consent was obtained from all patients before specimen collection.
Reagents
The H3K4 methyltransferase LSD1 inhibitor S2101 was pur- chased from Merck Millipore (Burlington, MA, USA). The LSD1 inhibitor GSK2879552, H3K27 methyltransferase EZH2 inhibitor GSK126, and H3K79 methyltransferase DOT1L inhibitor EPZ- 5676 were all purchased from Chemietek (Indianapolis, IN, USA). The H3K9 methyltransferase G9a inhibitor UNC0638, H3K27 demethylase JMJD3/UTX inhibitor GSKJ1, DOT1L inhibitor SGC0946, and MYC inhibitor 10058-F4 were all purchased from Sigma-Aldrich (St. Louis, MO, USA).
Drug treatment and cell viability assay
To screen for anti-proliferative effects of histone methyltrans- ferase or demethylase inhibitors, MM cell lines (3×104 to 1×105 cells/well in 6-well plate) were treated with the respective drugs at a concentration of 1 mM or with DMSO for up to 14 days, refresh- ing the medium and drugs every 3 to 4 days. Cell viabilities were assessed on days 3-4 and 11-14 using a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) and a microplate reader (Model 680; Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. To further analyze the effect of DOT1L inhibitors, MM cell lines (2×104 to 8×104 cells/well in 6-well plate) or patient- derived CD138-positive cells (1.3×105 to 3×105 cells/well in 6-well
plate) were treated with the respective inhibitors at 0.25-1 mM or with DMSO for up to 18 days, refreshing the medium and drug every 3 days.
Xenograft studies
For xenograft studies, we used the ex vivo drug pre-treatment method.15,16 RPMI-8226 cells were pre-treated for 3 days with 1 mM SGC0946 or EPZ-5676 or with DMSO, after which 1×107 cells were suspended in 200 ml of RPMI-1640 medium and subcuta- neously injected into the bilateral thighs of 6-week-old C.B-17 SCID mice. Tumor size was measured every 3 days using digital calipers, and tumor volume was calculated using the formula, length × width2/2. All animal experiments were conducted in compliance with the protocol approved by the Institutional Animal Care and Use Committee of Sapporo Medical University.
Results
DOT1L is a potential therapeutic target in MM
To determine whether histone methylation modifiers could be useful therapeutic targets in MM, we first tested the effects of the following compounds on proliferation of MM cell lines: the LSD1 inhibitors S2101 and GSK2879552, the G9a inhibitor UNC0638, the EZH2 inhibitor GSK126, the JMJD3 inhibitor GSKJ1 and the DOT1L inhibitor SGC0946 (Figure 1A). Five MM cell lines were treated with the drugs (1 μM) for up to 14 days, and cell viabilities were assessed early (days 3-4) and late (days 11-14) during the treatment. We found that inhibitors of G9a, EZH2 and DOT1L each moderately suppressed proliferation of more than 2 MM cell lines at the early times (Figure 1A). Longer treatment with these drugs led to stronger growth suppressive effects in most of the MM cell lines, though not in KMS-12PE cells (Figure 1A). By contrast, the LSD1 inhibitors were less effective. We tested 2 LSD1 inhibitors (S2101 for KMS- 12BM and MM.1S cells and GSK2879552 for RPMI-8226, KMS-12PE and U-266 cells), but neither suppressed MM cell proliferation and appeared to even promote it in most cases (Figure 1A). Among them, we selected DOT1L for further analysis, because its inhibition had a strong anti- proliferative effect.
Analysis using published data sets revealed that expres- sion of DOT1L is increased during the progression from normal plasma cells (NPCs) to monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SmMM) (Figure 1B). By contrast, we detected no significant difference in the levels of DOT1L expression between SmMM and symptomatic MM (SyMM) (Figure 1B). qRT-PCR showed that DOT1L is expressed at various levels in the MM cell lines tested, irrespective of their sensitivity to the DOT1L inhibitor (Figure 1C).
DOT1L inhibitors induce growth suppression, cell cycle arrest and apoptosis in MM cells
To further evaluate the therapeutic potential of DOT1L inhibition in MM, we treated MM cell lines with two DOT1L inhibitors, SGC0946 and EPZ-5676. Western blot analysis using an antibody specific for mono-, di-, and trimethylated H3K79 (H3K79me1/me2/me3) showed that treatment with either drug (1 mM, 3 days) significantly reduced levels of H3K79me1/me2/me3 in RPMI-8226 cells (Online
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