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CHD4 is required for maintenance of childhood acute myeloid leukemia
we performed RNA-Seq analysis of human THP-1 AML cells transduced with shRNAs targeting CHD4.
Density and boxplot analysis of the samples illustrated an even distribution of counts per million (CPM) after normalization and clustering analysis (Online Supplementary Figure S7A,B). Heatmap clustering of the top 100 upregulated or downregulated genes (Figure 7A) of the RNA-Seq data showed a high degree of repro- ducibility between the triplicates, and a significant differ- entiation between the AML CHD4 knockdown cells and the cells transduced with a scramble control.
Differentially expressed genes were those in which the mRNA levels were changed >1 or log2 fold change <-1; P<0.05; false discovery rate (FDR) <0.05 (Online Supplementary Table S4). Consistent with a role in gene repression,8 a majority of the genes were found to be upregulated upon knockdown of CHD4 (1011 genes were upregulated whereas 413 genes were downregulated). CHD4 and some of its previously reported target genes (e.g., IGF2, TGFB1 and PDGFA)38 were among those with the most significant changes in mRNA levels (Online Supplementary Table S4).
Gene set enrichment analysis (GSEA) was used to investigate whether the transcriptome profile generated by the inhibition of CHD4 was associated to the collec- tion of annotated gene sets (i.e., The Molecular Signatures Database [MSigDB]).43 The Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis revealed that genes deregulated upon CHD4 suppression were enriched for gene sets such as spliceosome, proteasome and base excision repair (Figure 7B). Intriguingly, the expression changes in response to the inhibition of CHD4 compared to the Molecular Signatures Database (MSigDB) hallmark gene sets were most significantly cor- related with the gene signature of two individual sub- groups of MYC targets data sets (FDR Q-value=0.0 and normalized enrichment score [NES]=-3.3; FDR Q- value=0.0, NES=-2.9, respectively), and to the MYC tar- get E2F transcription factor (E2F) and its target genes with cell cycle related functions (FDR Q-value=0.0, NES=-2.6) (Figure 7C-E). Consistent with the link to MYC and its important role in cell cycle regulation, GSEA analysis using the reactome gene sets and comparisons to changes in gene expression upon CHD4 knockdown revealed sig- nificant associations with several gene sets involving cell cycle progression, including S phase (NES=-2.7; FDR Q=0), synthesis of DNA (NES=-3.3; FDR Q=6.64E-04) and assembly of the pre-replicative complex (NES=-2.4; FDR Q=0.00162) (Figure 7F-H).
Further analysis of the RNA-Seq data revealed that inhibition of CHD4 caused decreased mRNA levels of MYC and several of its target genes involved in G1/S cell cycle transition, including cyclin D1, D2, E1, E2F1, and E2F2. Conversely, the negative cell cycle regulator p27 was shown to be upregulated. In contrast, other MYC tar- gets with alternative roles in other stages of the cell cycle, such as cyclin A2, B1, E2, displayed less pronounced changes in mRNA levels (Online Supplementary Table S4). To validate the RNA-Seq findings, we performed an addi- tional shRNA-based knockdown of CHD4 in THP-1 cells. qPCR analysis confirmed the RNA-Seq data, but with even more significant changes in the gene expression of MYC and an asset of its targets genes known to be involved in cell cycle progression (Figure 7I). Consistent with this, an additional RNA-Seq analysis of THP-1 cells
transduced with an independent shRNA against CHD4 again showed significant correlations to MYC and E2F targets. In addition, qPCR-based validation of the RNA- Seq data confirmed the previous results in Figure 7I and the observed deregulation of MYC and its downstream targets (Online Supplementary Table S4; Online Supplementary Figure S8). Thus, inhibition of CHD4 was significantly associated with MYC targets and gene sets involved in S phase cell cycle progression.
Discussion
In this study, we performed loss of function screens on a large scale in AML cells and non-transformed BMs. We identified the epigenetic factor CHD4 as being essential for maintenance of LICs and disease progression of child- hood AML, but not for normal hematopoietic cells. CHD4 inhibition in AML cells caused downregulation of MYC and its target genes and an arrest in the G0 phase of cell cycle progression.
It is of utmost importance that future treatments for AML selectively target the cancer cells without harming normal cells. Accordingly, we showed that CHD4 is required for cell growth of leukemic cells carrying various genetic lesions and for disease progression using an immune competent mouse model. However, CHD4 is not essential for primary normal murine BMs or for nor- mal human UCBs. Our findings are supported by previ- ous reports showing that inhibition of CHD4 is not cru- cial for normal hematopoietic cell growth,14 but has nonessential functions in self-renewal and lineage choice in normal hematopoiesis.44,45 Interestingly, this selectivity seems to be conserved in breast cancer.9 Indeed, CHD4 has previously been demonstrated to be required for growth of a broad range of cancer cells,9,13,37,38,46,47 including colony formation capacity of AML cells,14 implying that CHD4 may represent a cancer-specific dependency in a wider repertoire of tumors.
Most importantly, our results highlight a novel and essential role for CHD4 in maintenance of childhood AML in vitro and in vivo. The use of appropriate co-culture systems and a patient-derived xenograft mouse model for childhood AML allowed us to demonstrate that the essential role of CHD4 was consistently manifested in patient samples carrying diverse types of genetic lesions as well as the LICs. Intriguingly, the importance of CHD4 in cancer-initiating cells has also been reported in hepato- cellular carcinoma47 and glioblastoma,38 indicating that the role of CHD4 in these cells that drive tumor growth may be more general than previously anticipated.
CHD4/NuRD has been shown to control cell cycle pro- gression in a p53 dependent manner,10-12 or in a p53 inde- pendent manner,9,48 and inhibition of CHD4 was shown to cause a cell cycle arrest in G1/S.11 In the present study, inhibition of CHD4 resulted in repression of MYC and its target genes involved in cell cycle progression and conse- quently caused a G0 cell cycle arrest. In support of this, inhibition of MYC has been reported to cause a G0/G1 block in the cell cycle in human lymphoid and myeloid cells.49 Moreover, CHD4 has been found to directly bind to the MYC promoter in glioblastoma cells and inhibition of CHD4 resulted in a downregulation of MYC.38 In addi- tion, MYC was also part of a set of genes suggested to have a role in colony formation in AML cells.14
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