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DOT1L is a therapeutic target in myeloma
expression of a number of genes in MM cells. Microarray analysis showed that 1255 probe sets were upregulated (> 1.5-fold) by SGC0946 in RPMI-8226 cells, as were 492 probe sets in MM.1S cells (Online Supplementary Tables S5 and S6). Among them, 143 probe sets (including 125 pro- tein coding genes) were upregulated in both cell lines (Online Supplementary Figure S8A). Gene ontology (GO) analysis suggested that genes involved in “immune sys- tem” and “immune response” were significantly enriched among the upregulated genes in both cell lines (Figure 5A), while pathway analysis revealed that genes associated with “interferon (IFN) signaling” were significantly enriched among the upregulated genes (Figure 5B). qRT- PCR analysis confirmed that a series of IFN-stimulated genes were upregulated by SGC0946 and EPZ-5676 in MM cells (Figure 5C). However, ChIP-seq analysis showed that H3K79me2 levels were not significantly altered at these genes (Online Supplementary Figure S8B). This suggests DOT1L inhibitors may affect immune responses and IFN signaling through a H3K79me2-inde- pendent mechanism in MM cells.
Gene mutations are potentially associated with the DOT1L sensitivity in MM cells
Although the KMS-12BM and KMS-12PE cell lines were established from the same patient,19 KMS-12PE cells were less sensitive to DOT1L inhibitors and other epigenetic drugs than KMS-12BM cells (Figures 1A and 2A). To deter- mine the molecular mechanism underlying this difference in drug sensitivity, we carried out targeted sequencing of a panel of cancer-related genes in these cell lines. In the 409 genes analyzed, mutations were detected in 12 genes in KMS-12BM cells, while 14 mutations in 13 genes were detected in KMS-12PE cells (Figure 6A, Online Supplementary Table S7). Among these mutations, 8 were found in both cell lines (Figure 6A). Notably, KMS-12PE cells exhibited mutations in multiple histone modifier genes (EP300, KMT2C and KMT2D), but KMS-12BM cells did not (Figure 6A).
Extended treatment enhances the effect of DOT1L inhibitors in MM cells
We next clarified whether IRF4-MYC signaling is asso- ciated with the different sensitivities of KMS-12BM and KMS-12PE cells to DOT1L inhibitors. qRT-PCR revealed that MYC and IRF4 were expressed at lower levels in KMS-12PE than KMS-12BM cells, suggesting lower IRF4- MYC signaling may be associated with the impaired anti- tumor effect of DOT1L inhibitors (Figure 6B). Consistent with that idea, KMS-12PE cells were also less sensitive to the MYC inhibitor 10058-F4 than were KMS-12BM cells (Figure 6C). By contrast, RPMI-8226 cells, which were sensitive to DOT1L inhibitors, were also highly sensitive to 10058-F4 (Online Supplementary Figure S9).
It is noteworthy, however, that DOT1L inhibitors sup- pressed expression of MYC and IRF4 in KMS-12PE cells (Figure 6D). Moreover, extended treatment with DOT1L inhibitors for up to 18 days led to strong growth suppres- sion in the less sensitive KMS-12PE and U-266 cell lines (Figure 6E). To clarify the mechanism underlying antitu- mor effect of the extended treatment, we performed gene expression microarray analysis with KMS-12PE cells treat- ed with SGC0946 or DMSO for 12 days. This analysis identified 509 (401 unique genes) probe sets that were downregulated (> 1.5-fold) and 865 (739 unique genes)
that were upregulated (> 1.5-fold) by SGC0946 in KMS- 12PE cells (Online Supplementary Tables S8 and S9). Among them, multiple IRF4-MYC signaling genes were downreg- ulated by SGC0946 (Figure 6F). Moreover, gene ontology and pathway analyses showed that genes associated with “immune response” and “IFN signaling” were significantly enriched among the upregulated genes (Figure 6G and H). These results demonstrate that the time required for DOT1L inhibitors to exert their effects varies among MM cells.
Discussion
In the present study, we show that DOT1L inhibitors exerted a strong anti-myeloma effect. Expression of DOT1L is significantly higher in SmMM than NPC, sug- gesting DOT1L may be causally associated with myelo- magenesis.
DOT1L is the only known histone methyltransferase that catalyzes mono-, di- and trimethylation at H3K79.20,21 In mammals, most of H3K79 is unmethylated, and H3K79 methylation is linked to active transcription.20,22 DOT1L is a component of large transcription complexes that also include transcription factors such as AF4, AF9, AF10, ENL and P-TEFb.20,23-26 Within these complexes, DOT1L may initiate or sustain active transcription by mediating H3K79 methylation. DOT1L is also a potential drug target in mixed lineage leukemia (MLL) gene rearranged leukemia. DOT1L forms a complex with MLL fusion proteins, and DOT1L-mediated H3K79 methylation leads to enhanced expression of target oncogenes, including HOXA9 and MEIS1.26,27 Recent studies have demonstrated the selective and strong antitumor effects of DOT1L inhibitors against MLL-rearranged leukemia.17,18,28 Similarly, DOT1L is a potential therapeutic target in lung and breast cancer with high DOT1L expression and neuroblastoma with MYCN amplification.29-31
We found that DOT1L inhibition targets the IRF4-MYC axis in MM cells (Figure 6I). Aberrant activation of several transcription factors, including MYC, MAF, NF-κB and IRF4, is involved in the development of MM.4 MM cell survival is strongly dependent on IRF4 and MYC, and MYC is a direct target gene of IRF4 transactivation, while IRF4 is a direct target of MYC.32,33 The IRF4-MYC axis is thus considered to be an important therapeutic target in MM, and a recent study showed that CBP/EP300 bromod- omain inhibitors directly suppress IRF4 expression and inhibit MM cell viability.34 Moreover, dependence on the KDM3A-KLF2-IRF4 axis was recently reported in MM.35 KDM3A maintains KLF2 and IRF4 expression via H3K9 demethylation, and KLF2 directly targets IRF4 while IRF4 reciprocally activates KLF2, forming a positive autoregula- tory circuit.35 We found that DOT1L inhibition leads to decreased levels of H3K79me2 and repression of IRF4 and its target genes, including MYC, PRDM1 (also known as BLIMP1) and KLF2 in MM cells.32 As previously shown, H3K79me2 peaked just behind the transcriptional start site of the active genes and gradually declined over the course of the gene body,20,36 and it was significantly deplet- ed in MM cells treated with a DOT1L inhibitor.
We found that genes associated with immune responses and IFN signaling were significantly upregulated by DOT1L inhibition in MM cells. The potential of IFN in the clinical treatment of MM has long been recognized.
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