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CHD4 is required for maintenance of childhood acute myeloid leukemia
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 mediated loss of function of CHD4 prevents cell growth of AML cells
To exclude the possibility that the observed effects of the CHD4 inhibition was caused by off target effects, we took advantage of the highly specific CRISPR-Cas9 genome editing technology.41 Accordingly, we generated MV4-11 and THP-1 AML cells constitutively expressing Cas9, and transduced the cells with two individual inducible vectors expressing guide RNA (gRNA) homolo- gous to the CHD4 locus (Figure 4A). Induction of expres- sion of both gRNAs resulted in a significant disruption of the CHD4 coding region (Figure 4B) and reduction of pro- tein levels (Figure 4C), in THP-1 AML cells. Although the reduction in protein levels in some experiments were rel- atively moderate, the specific CRISPR-Cas9 targeting of CHD4 caused a marked reduction in live THP-1 cells, confirming the essential role of CHD4 in AML cell growth (Figure 4D,E). Comparable results were obtained using the same approach with the human MV4-11 AML cells (Figure 4F-I) and the mouse MLL-AF9 AML cells (Online Supplementary Figure S4), thereby further validat- ing that CHD4 is required for AML cell growth.
CHD4 suppression impairs maintenance of primary childhood AML cells ex vivo
We demonstrated that CHD4 inhibition prevented growth of childhood AML cell lines (Figure 2 and Figure 4). Thus, we next investigated the importance of the inhi- bition of CHD4 in a cohort of childhood AML patient samples (Online Supplementary Table S3). To enable main- tenance of the primary human childhood AML cells with high viability, including LICs, we utilized a stromal cell co-culture system for the cell growth assays.28 shRNA- mediated knockdown of CHD4 using two independent constructs proved to be efficient for four out of six sam- ples from which we could extract good quality mRNA (Figure 5A; Online Supplementary Figure S5A), and indeed from one sample we had enough material for western blot analysis (Online Supplementary Figure S5B). Flow cyto- metric analysis showed that leukocytes (CD45+ cells) and LICs (Lin-CD34+CD38– cells) of primary childhood AML samples transduced with a negative control vector could be maintained on stromal feeder layers with high viabili- ty (80-90%) up to five weeks after transduction (Figure 5B-D; Online Supplementary Figure S5C,D).
In contrast, shRNA-mediated targeting of CHD4 in a primary childhood sample carrying the MLL translocation (Online Supplementary Table S3, sample “AML 6”) prevent- ed maintenance of leukocytes and LICs compared to the control sample (Figure 5C,D). We then sought to investi- gate if CHD4 was required for maintenance of childhood AML samples carrying alternative genetic lesions (Online Supplementary Table S3). With the exception of LICs in one patient sample (sample “AML 2”, Figure 5D), the sup- pression of CHD4 with two independent shRNAs pre- vented the maintenance of leukocytes and LICs in all of the tested non-MLL rearranged childhood AML samples compared to the control cells (Figure 5B-D; Online Supplementary Figure S5C,D). Although all non-MLL rearranged samples did not robustly expand, the suppres- sion of maintenance was similar to that of the MLL- rearranged primary childhood AML sample (“AML 6”) (Figure 5B-D; Online Supplementary Figure S5C,D). These results show that the inhibition of CHD4 prevents
growth of bulk cancer cells/leukocytes and LICs of pri- mary childhood AML BMs with different genetic lesions.
CHD4 suppression induces anti-leukemic effects in primary childhood AML cells in vivo
To investigate whether CHD4 also was required for growth of primary childhood AML cells in vivo, we used a humanized NSG-SGM3.42 Patient sample “AML 2” and “AML 7” (Online Supplementary Table S3), transduced with CHD4 targeting shRNA vectors or control vectors (Figure 5E), were intrafemurally transplanted into recipient mice. NSG-SGM3 mice transplanted with childhood AML cells transduced with control vectors resulted in a significant level of engraftment for both transplanted patient sam- ples (median value of 5.7% +/- 2, and 9.6% +/- 2), deter- mined by flow cytometric analysis of CD45+ cells, eight weeks post transplantation (Figure 5F). In sharp contrast, primary childhood AML cells transduced with shRNA vectors efficiently knocking down CHD4 mRNA (Figure 5E) displayed significantly lower levels of leukemic cell engraftment compared to the control cells (median value of 1.6, 0.03, P=0.0152, P<0.001, respectively) (Figure 5F). Thus, the targeting of CHD4 inhibits cell proliferation of primary childhood AML cells and AML progression in vivo.
CHD4 controls cell cycle progression of AML cells
To investigate the cellular mechanisms by which CHD4 was required for AML cell growth in childhood AML, we used two MLL rearranged (THP-1 and MV4-11) and two non-MLL rearranged (AML-193 and Kasumi-1) childhood AML cell lines, and analyzed the effects in apoptosis and the cell cycle upon suppression of CHD4 expression. shRNA-mediated inhibition of CHD4 resulted in a signif- icant reduction of mRNA (Figure 2A and Figure 6A) and protein levels (Figure 2B and Figure 6B), and a dramatic accumulation of all four cell lines in the G0 phase of the cell cycle, compared to the control cells. In addition, all cell lines displayed a decrease of cells in G1, whereas less pronounced effects was observed in the S and G2/M phase, compared to the control cells (Figure 6C-G). The robust arrest in G0 when CHD4 was inhibited (Online Supplementary Figure S3A,B; Online Supplementary Figure S6A, B), was confirmed in all four cell lines using an inde- pendent shRNA against CHD4 (Online Supplementary
Figure S6C-F).
In contrast to the strong effects in cell cycle progression,
CHD4 depleted cells (Figure 2A,B and Figure 6A,B) dis- played modest levels of early apoptotic cells (Annexin V+NIR–), and late apoptotic cells (Annexin V+NIR+) (Figure 6G-J). Likewise, knockdown of CHD4 using an independ- ent shRNA (Online Supplementary Figure S3A,B and Online Supplementary Figure S6A,B) resulted in the same relatively low levels of apoptosis in all cell lines (Online Supplementary Figure S6G-J). Taken together, these data indicate that the primary cellular mechanism, whereby inhibition of CHD4 prevented cell growth of AML cells, is mediated via a growth arrest of the cells in the G0 phase of cycle progression, rather than induction of apop- tosis.
CHD4 suppression induces an expression profile correlating to MYC targets
To delineate the underlying molecular mechanisms whereby CHD4 is required for the growth of AML cells,
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