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The chromodomain helicase DNA-binding protein 4 (CHD4) is an adenosine triphosphate (ATP)-ase depend- ent chromatin remodeling factor and a component of the nucleosome remodeling and histone deacetylation (NuRD) complex and plays an important role in epigenet- ic transcriptional gene regulation.8 CHD4 has been linked to oncogenic processes, including control of cell cycle pro- gression,9-12 cancer metastasis, epithelial-to-mesenchymal transition,13 and epigenetic suppression of tumor suppres- sor genes.13 Although the role of CHD4 in AML is largely unknown, inhibition of the chromatin remodeler has been reported to reduce AML tumor formation and sensi- tize AML cells to genotoxic drugs via the increased acces- sibility of DNA and impaired double strand break repair.14
A large number of functional screens have identified essential genes in various cancer cells,15 including AML.16- 27 However, AML-specific vulnerabilities have not been studied in detail. In the study herein, we performed loss of function screens on a large scale in AML cells and non- transformed bone marrow cells (BMs) in order to identify potential AML-specific vulnerabilities. CHD4 was identi- fied as being required for cell growth and disease progres- sion for primary childhood AML patient samples, but not for primary blood cells. Inhibition of CHD4 resulted in a downregulation of MYC and its target genes as well as a growth arrest in the G0 phase of the cell cycle.
Methods
Cell growth assays of primary childhood AML samples
The investigation was conducted in accordance with the ethi- cal standards and according to the Declaration of Helsinki and to national and international guidelines, and has been approved by the authors' institutional review board. Culturing of the child- hood samples was carried out as previously reported.28 MS-5 cells (DSMZ) were radiated at 80 Gy and plated at a density of 10,000 cells/well in MyeloCult media H5100 (STEMCELL Technologies Inc.) in a collagen I Cellware 96-well plate (Corning), two to three days before plating the cells. 10,000-20,000 cells suspended in MyeloCult media supplemented with recombinant human inter- leukin-6 (rhIL-6), recombinant human interleukin-3 (rhIL-3), recombinant human Fms-like tyrosine kinase 3/fetal liver kinase- 2 (rhFl3/Flk-2) ligand, recombinant human thrombopoietin (rhTPO), recombinant human stem-cell factor (rhSCF) and recombinant human granulocyte colony-stimulating factor (rhG- CSF; STEMCELL Technologies Inc.) at a concentration of 20 ng/mL, were added to each well. The cells were maintained at normoxic conditions and effects in cell growth (LICs and leuco- cytes) were determined by flow cytometric analysis (see Online Supplementary Table S1 for antibodies).
Flow cytometric analysis and sorting
Flow cytometric analysis was performed with a 4-laser BD LSRFortessa. Primary childhood AML cells were harvested and incubated in anti-CD16/32 (Fc-block) antibodies against mouse (Biolegend) and human (ChromPure Mouse IgG, Jackson ImmmunoResearch) for 20 minutes on ice. Then, the cells were stained with: human CD45, CD34, CD38 and lineage antibodies (CD20, CD4, CD8, CD2, CD56, CD235b, CD3 and CD19) and incubated on ice for 20 minutes (see Online Supplementary Table S1 for antibodies). Dead cells were excluded using the Near-IR Live/Dead marker (Invitrogen). Human CD45 positive cells were analyzed by a high-throughput automated plate reader (BD LSRFortessa).
For the cell growth competition assays, cells were harvested and washed with cold phosphate-buffered saline (PBS) and thereafter stained with Near-IR Live/Dead marker in a 96-well plate in 80 ml of PBS and 2% fetal bovine serum (FBS). A high- throughput automated plate reader was used to detect the absolute number of live cells.
To determine the level of engraftment of human AML cells in transplanted NOD scid g mice expressing human SCF, GM-CSF, and IL-3 (NSG-SGM3), BMs were isolated from the tibia and femur. The isolated BMs were incubated in mouse and human FC blocking antibodies for 20 minutes on ice. After washing, the cells were stained with human anti-CD45 on ice for 20 minutes. Then cells were incubated with Near-IR Live/Dead marker to detect live cells. Analysis was performed by FlowJo Version 9.3.3 software (Tree Star Inc.).
Results
Identification of target genes selectively required for growth of MLL-AF9 rearranged AML cells by large-scale short hairpin RNA (shRNA) screening
To identify novel target genes that are required for growth of AML cells expressing the MLL-AF9 fusion oncogene, we performed shRNA-based screens of two human AML cell lines (NOMO-1 cells derived from an adult, and THP-1 cells derived from a 1-year-old child) and a mouse AML cell line, all of which carried the MLL- AF9 translocation. Non-transformed mouse Factor- Dependent Continuous Paterson Laboratories (Factor dependent cell-Paterson [FDCP]-mix) BMs were used as control cells in the screens.29 The RNA interference (RNAi) screening systems consisted of approximately 27000 lentiviral shRNAs targeting around 5400 putative disease-associated and drug targets (Cellecta Inc.).
As outlined in Figure 1A, the barcoded lentiviral libraries were transduced as a pool into respective cell lines. The cells were harvested at an initial time point and after ten cell divisions. Next-generation sequencing of the polymerase chain reaction (NGS PCR) amplified barcodes from genomic DNA was followed by deconvolution and normalization of the data (Online Supplementary Table S2).
To identify target genes that were selectively required for AML cell growth, we first determined the ratio of each individual barcode in the mouse AML cells com- pared to the mouse FDCP-mix cells after ten cell divi- sions. We identified 1082 target genes with at least a five- fold higher effect in cell growth in the mouse AML cells compared to the FDCP-mix cells (Figure 1B). By compar- ing these 1082 target genes with the corresponding human target genes of the human THP-1 and NOMO-1 cells (Figure 1C,D), we identified 34 shRNA-target genes that overlapped with all three screens (Figure 1E). Notably, 201 of the 289 total target genes from the screens of the two human AML cell lines overlapped (P=2.4E-215, hypergeometric test), indicating a conserved functional importance of these genes in AML mainte- nance (Figure 1E).
Although most of the identified target genes had not been reported to have a role in AML, some have previ- ously been linked to the disease, including MED12,30 USP7,31 FIP1L1,32 and SMC1A.33 Additionally, a significant proportion of the genes (15 of 34) were targeted by mul- tiple shRNAs (Figure 1F). Gene ontology analysis revealed that the 34 target genes were associated with a broad
haematologica | 2018; 103(7)