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CHD4 is required for maintenance of childhood acute myeloid leukemia
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Figure 5. CHD4 inhibition prevents growth of patient-derived childhood AML cells ex vivo and in vivo. A. Bar charts show real time PCR analysis of mRNA levels after shRNA-based knockdown of CHD4, relative to control cells transduced with vectors expressing scrambled shRNA, of the indicated primary childhood AML samples used in the proliferation assays (Figure 5B-D), at 72 hours post transduction. mRNA levels were normalized to UBC. The data is represented as the mean ±S.E.M., **P<0.01, ***P<0.005, ****P<0.001 (unpaired t-test), n=3. B. Flow cytometry charts of expression profiles of CD34, CD38, Lin, and CD45 of cells transduced with CHD4 shRNA expressing RFP (CHD4-shRNA) or a negative control vector expressing scrambled shRNA and GFP (Control shRNA). The patient-samples were co- cultured with murine MS-5 stromal cells until the control cells reached confluence, or up to five weeks of maintenance. The number of viable CD45+ or Lin- CD34+CD38–, were determined by flow cytometric analysis. Live cell populations negative for Live/dead Near-IR dye was used for flow cytometric analysis. The num- bers in each square represent the percentage of cells within each cell population. C and D. Bar chart showing the total numbers of live CD45+ (Figure C) and Lin- CD34+CD38– (Figure D) primary childhood AML cells transduced with CHD4 shRNA, or a control vector, as indicated. *P<0.05, **P<0.01, ***P<0.005, ****P<0.001 (unpaired t-test). E. Bar charts depict real time PCR analysis of mRNA levels after shRNA-based knockdown of CHD4, relative to control cells trans- duced with vectors expressing scrambled shRNA, of the indicated primary childhood AML samples used for the xenograft experiments (Figure F) at 72 hours post transduction. mRNA levels were normalized to UBC. The data is represented as the mean ±S.E.M., **P<0.01 (unpaired t-test), n=3. F. Scatter plot of percent of engrafted primary childhood AML cells of two childhood patient samples, transduced with shRNA against CHD4, or a negative control vector, that were transplanted into humanized NSG-SGM3 recipient mice. Each animal was transplanted by intrafemoral injection with a single cell dose of up to 300,000 unfractionated primary cells. The childhood AML BMs were harvested after eight weeks post transplantation and the level of engraftment was determined by flow cytometric analysis. Each symbol represents an individual transplanted mouse, and each sample was transplanted in triplicates. *P<0.05, ****P<0.001 (unpaired t-test). Sc: scramble con- trol; shRNA: short hairpin RNA; ns: non-significant; AML: acute myeloid leukemia.
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