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DOT1L is a therapeutic target in myeloma
Supplementary Figure S1) and strongly suppressed prolifer- ation of RPMI-8226, MM.1S, KMS-11 and KMS-12BM cells (Figure 2A). That the drugs were acting through DOT1L inhibition was confirmed by the finding that shRNA-mediated DOT1L knockdown moderately sup- pressed MM cell proliferation (Online Supplementary Figure S2). On the other hand, DOT1L inhibitors were less effective or ineffective in KMS-12PE and U-266 cells (Figure 2A). Moreover, ex vivo treatment with SGC0946 or EPZ-5676 strongly suppressed tumor formation by MM cells in SCID mice (Figure 2B, Online Supplementary Figure S3). To evaluate the effects of DOT1L inhibitors in
primary tumors, we isolated CD138-positive cells from MM and PCL patients. We found that both drugs moder- ately suppressed the viability of primary tumor cells (Figure 2C).
Cell cycle analysis using flow cytometry revealed that treatment with SGC0946 (1 mM, 6 days) or EPZ-5676 (1 mM, 6 days) led to increases in sub-G1 and G0-G1 phase populations and decreases in S phase populations in RPMI-8226 and MM.1S cells, which suggests DOT1L inhibition induces G1-S arrest and apoptosis (Figure 3A). Induction of apoptosis by DOT1L inhibitors was con- firmed using Annexin V staining assays (Figure 3B, Online
A
Figure 1. Identification of DOT1L as a potential therapeutic target in MM. (A) Effects of inhibitors of histone methylation modifiers on MM cell prolifera- tion. Shown are summarized results of cell viability
BC assays in MM cell lines treated with the indicated drugs (1 mM) at early and late times. Results are normalized to cells treated with DMSO. The data are presented as means of 5 replications; error bars represent standard errors of means (SEMs). Statistical analyses compared cells treated with DMSO and those treated with the indicated drugs using t-tests (unpaired, two-sided). * P<0.05. (B) Comparison of DOT1L mRNA expression among normal plasma cells (NPC, n=22), monoclonal gam- mopathy of undetermined significance (MGUS, n=44), and smoldering multiple myeloma (SmMM, n=12) (left) and between SmMM (n=24) and symp- tomatic MM (SyMM, n=69) (right) using the indicat- ed datasets. (C) qRT-PCR analysis of DOT1L in the indicated MM cell lines. Results are normalized to ACTB expression. Shown are means of 3 replica- tions; error bars represent standard errors of
means (SEMs).
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