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J. Ropa et al.
untreated control (Online Supplementary Figure S4D-E). This may suggest Setdb1 is maintained within a narrow expression window in AML cells.
To test the effect of Setdb1 knock-down on HSPC colony formation we transduced Lin– bone marrow with shRNA targeting Setdb1. Our results show a trend for increased HSPC colony formation without changes in Hoxa9 or Meis1 expression following Setdb1 knockdown (Online Supplementary Figure S4F, data not shown). Because inhibition of H3K9 methyltransferases has been proposed as a therapeutic option to treat AML,22,23 we explored the effects of chemical inhibition of H3K9 methylation in nor- mal and malignant hematopoietic cells. Without a selec- tive SETDB1 small molecule inhibitor, we utilized the G9a inhibitor UNC0638, which addresses the function of H3K9 methyltransferases more broadly. Lin– mouse bone marrow cells treated with UNC0638 exhibit a reduction in both H3K9me2 and H3K9me3 (Figure 3A).28,29 Given our data demonstrating Hoxa9 and Meis1 are repressed by H3K9 methylation,5 we tested the effect of UNC0638 on HSPC self-renewal. Previous reports have demonstrated that mouse Lin–Ska1+cKit+ cells (LSK) are preserved in cul- ture following treatment with UNC0638.30 To explore this further, Lin– cells were treated with increasing doses of UNC0638 for five days prior to plating in semi-solid methylcellulose in the presence of SCF and IL-3. Interestingly, treatment with UNC0638 significantly increased colony formation capacity of Lin– cells in a dose- dependent manner (Figure 3B-C). Further, human CD34+ cells isolated from mobilized peripheral blood and treated with increasing doses of UNC0638 demonstrate increased colony formation capacity (Figure 3D-E), consistent with reports that UNC0638 preserves CD34+ cells in culture.31 Thus, chemical or genetic inhibition of H3K9 methyltrans- ferases preserves self-renewal capacity of bone marrow cells.
We also examined the effects of UNC0638 chemical inhibition of H3K9 methylation on AML cells. Consistent with previous studies, treatment with UNC0638 results in reduced cellular proliferation of MLL-AF9 cells (Online Supplementary Figure S5A).22 Since UNC0638 preserves self-renewal of HSPC (Figure 3), we asked whether inhibi- tion of H3K9me alters MLL-AF9 mediated transformation of bone marrow cells. First, we found that UNC0638 treat- ment of Lin– bone marrow cells preserve more primitive cKit+ and Cd11b– populations (Figure 4A-D and Online Supplementary Figure S5B-C). Lin– cells display significantly increased Hoxa9 expression in response to UNC0638 treatment (Figure 4E). Lin–cKit+ (LK) and related primitive hematopoietic cell populations are more amenable to transformation than more differentiated subtypes.32 To explore whether this expansion of LK cells results in greater AML transformation capacity, we pretreated Lin– bone marrow cells with UNC0638 for four days then retrovirally transduced them with MigR1-MLL-AF9 and monitored green fluorescent protein (GFP) by flow cytom- etry. We observed a more rapid expansion of GFP+ MLL- AF9 cells following treatment with UNC0638 compared to vehicle, with a 1.4-1.7 fold increase in GFP+ cells before both populations reached 100% GFP positivity (Figure 4F- G and Online Supplementary Figure S5D). One of four repli- cates demonstrated a more rapid expansion of vehicle treated cells; however, GFP+ cells were increased two-fold in vehicle treated cells at day 1 suggesting this is attribut- able to differences in transduction rates. Our combined
data suggests that H3K9 methyltransferases can suppress leukemic transformation and may point to a narrow win- dow of H3K9 methylation that is optimal for leukemic transformation and cell growth.
SETDB1 regulates oncogenic gene programs in AML
To explore the gene programs regulated by SETDB1 in AML, we performed RNA-sequencing experiments on MLL-AF9 cells overexpressing SETDB1. 2,285 genes are upregulated and 1,771 genes are downregulated by SETDB1 overexpression with a fold-change of 1.5 or more and an false discovery rate (FDR) of <0.05 (Figure 5A and Online Supplementary Table S4). Notably, many genes found in the Hoxa cluster were significantly downregulat- ed, including Hoxa3, Hoxa5, Hoxa6, Hoxa9, and the Hoxa9 cofactor Meis1; while only one Hoxa gene was upregulat- ed, the long non-coding RNA Hoxa11os (Figure 5A, high- lighted genes). In fact, gene programs that are upregulated by forced expression of HOXA9 and MEIS1 in mouse cells are significantly downregulated by SETDB1 overexpres- sion using gene set analysis33 (Figure 5B). We show signif- icant upregulation of genes that exhibit increased expres- sion in mature blood cells compared to HSPC, consistent with the differentiation observed upon SETDB1 overex- pression in AML34,35 (Figure 5C-D and Figure 2F-G). Because SETDB1 binds the PAF1c, which is required for localization of MLL fusion proteins,5 we asked how SETDB1 expression affects direct targets of MLL-AF9. Interestingly, genes bound by regulation of MLL-AF9 were significantly downregulated upon SETDB1 overexpres- sion,36 suggesting H3K9me3 regulation MLL-AF9 gene pro- grams in leukemic cells (Figure 5E). 193 genes downregu- lated by SETDB1 overexpression are reported to have reduced promoter H3K9 methylation in AML relative to normal CD34+ cells,17 suggesting SETDB1 may be respon- sible for regulating a subset of these genes, including Kit, Cbl, Ptpn11, Six1, and other genes that are important in AML (Figure 5F and Online Supplementary Table S4). There is significant overlap between genes downregulated by SETDB1 and genes upregulated by Crispr/Cas9 mediated knockdown of SETDB1 in human THP-1 AML cells har- boring an MLL-AF9 fusion (Figure 5E and Online Supplementary Table S4).24 This suggests SETDB1 regulates conserved pro-leukemic gene programs in leukemic cells
including direct MLL fusion targets.
SETDB1 regulates the epigenome to affect changes in chromatin accessibility and gene expression
We have shown that overexpression of SETDB1 in MLL-AF9 cells leads to global increases in H3K9me3.5 To understand the specific epigenomic changes induced by SETDB1 in AML, we performed ChIP-seq for H3K9me3 in MLL-AF9 and MLL-AF9+SETDB1 leukemic cells. We also performed ATAC-seq to assess changes in chromatin accessibility. We first explored differences at the HOXA locus due to its importance in a large subset of AML, including MLL leukemias.5,37 Overexpression of SETDB1 reduced chromatin accessibility and increased H3K9me3 at posterior Hoxa genes, which results in reduced tran- scription of Hoxa9 (Figure 6A). We sought to define whole genome epigenetic regulation mediated by SETDB1 but observed only 552 consensus H3K9me3 peaks (Online Supplementary Figure S7A and Online Supplementary Table S5), which included regions enriched for H3K9me3, such as the zinc finger protein cluster on chromosome 7 (Online
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