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J. Ropa et al.
Taken together, our data demonstrate profound effects of SETDB1 on the epigenome and transcriptome that affects genes critical for AML.
Discussion
Here we demonstrate that SETDB1 and H3K9 methyla- tion suppresses AML disease progression in vivo through the repression of pro-leukemic genes including direct MLL-fusion protein targets. We found that AML patient samples exhibit lower SETDB1 expression compared to normal hematopoietic cells and that higher SETDB1 expression correlates strongly with better overall AML patient survival (Figure 1). We recapitulated these findings in mice where forced expression of SETDB1 in MLL-AF9 driven AML induces differentiation of AML cells and increases disease latency (Figure 2). These data suggest SETDB1 suppresses AML cell growth and self-renewal by relieving the block in differentiation.
We attribute the phenotypes in AML cells to altered H3K9 methylation. We altered H3K9 methylation levels genetically (SETDB1 and G9a) and through small mole- cule inhibition (UNC0638). Similar to our results with SETDB1, manipulation of G9a suggests H3K9 methyla- tion can suppress AML progression by promoting differ- entiation (Figures 3-4, and Online Supplementary Figure S3). Thus, H3K9 methylation may have a more general effect on AML initiation and progression. Interestingly, Lehnertz and colleagues reported G9a overexpression accelerated Hoxa9/Meis1 mediated leukemia in vivo. We found Hoxa9/Meis1 mediated transformation in vitro was inhibit- ed by SETDB1, but to a lesser extent than MLL-AF9 (Figure 8). Unique experimental strategies or functions for SETDB1 and G9a may account for these differences.22 Consistent with a role for H3K9 methylation in suppress- ing hematopoietic transformation, deletion of the H3K9 methyltransferase, SUV39H1 (and to a lesser degree SUV39H2), leads to the development of B-cell lymphomas in mice.44 Additionally, SETDB2 resides in a region of chro- mosome 13 that is commonly deleted in chronic lympho- cytic leukemia (CLL).45 Thus, H3K9 methylation is likely exquisitely regulated in hematopoietic cells and performs context dependent functions that require further investi- gation to fully understand its role in AML.
Mechanistically, we found that SETDB1 is linked with altered H3K9 methylation and acetylation, decreased chromatin accessibility and transcriptional repression of critical AML oncogenes (Figures 5-7). These genes includ- ed several that have been implicated in myelodysplastic syndromes (MDS) and AML.39 We show SETDB1 regu- lates Dock1 expression, which is correlated with leukemic stem cell gene signatures and a poor prognosis in AML patients.41,46 We also observed that SETDB1 represses genes associated with AML, such as Hoxa9 and Six1, which are direct targets of MLL-AF9.16,37,40 Interestingly, Six1 was recently shown to be important for leukemic stem cell maintenance where depletion of Six1 leads to
increased disease latency.47 This suggests repression of Six1 may contribute to SETDB1 mediated extension of leukemic disease latency. These data point to SETDB1 negatively regulating a pro-leukemic gene program, many of which are potential therapeutic targets. Thus, under- standing the mechanisms regulating SETDB1 at the tran- scriptional and post-translational level may be a valuable therapeutic approach for AML. For example, miRNA29 is a critical mediator of SETDB1 expression.48 Another poten- tial mediator of H3K9 methylation is the PAF1c. We showed SETDB1 binds to the PAF1c and mediates pro- moter H3K9me3 of the Hoxa9 and Meis1 loci.5 Further, we and others identified G9a and SUV39H1 as interacting partners of the PAF1c.5,6 Interestingly, SETDB1, G9a, GLP and SUV39H1 form a complex that directs H3K9 methy- lation to euchromatic gene promoters.49 Thus, the PAF1c may recruit H3K9 methyltransferases to specific targets to mediate gene repression. The PAF1c is a critical regulator of transcription of several pro-leukemic genes in AML cells through direct physical interaction with wild-type MLL and MLL-fusion proteins.13,15 It will be interesting to consider the biochemical interplay between H3K9 methyltransferases and MLL-fusion proteins with the PAF1c.
Previous studies have demonstrated that SETDB1 and G9a are required for AML initiation and progression.22–24 Our current data demonstrating that SETDB1 suppresses AML growth may suggest AML cells maintain a narrow SETDB1 expression level. We show increased SETDB1 expression induces differentiation of AML cells through H3K9me3 and repression of self-renewal genes. Conversely, loss of SETDB1 is detrimental to leukemic cells due to derepression of endogenous retroviral ele- ments (ERV) and inhibition of HOXA9 transcriptional activity22,24 (Figure 8C). Given the essential role for SETDB1 in leukemia, small molecule inhibition of H3K9 methyltransferases has been proposed as a therapeutic option.22,23 However, a recent study shows depletion of G9a increased cancer progenitor cell populations that ini- tiate a delayed but more aggressive disease state.21 Thus, it is critical to fully understand the effects of chemically inhibiting of H3K9 methylation as a treatment for AML. Further investigation into the roles of SETDB1, G9a and more generally H3K9 methylation levels will likely shed light on the precise role of these methyltransferases in nor- mal and malignant hematopoiesis and determine the value of these epigenetic modifiers as therapeutic targets.
Acknowledgments
The authors thank Dr. Jianyong Shou for a SETDB1 expres- sion construct and Dr. Schahram Akbarian for Setdb1fl/fl mice. We also thank Dr. Russell Ryan, Dr. Emmalee Adelman, Dr. Maria Figueroa, and Dr. Sami Malek for helpful discussion.
Funding
This work was supported by NIH grants R01-HL-136420 (AGM), T32CA140044 (JR) and P30CA046592 (University of Michigan Flow Cytometry Core).
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