PU.1 is linking the glycolytic enzyme HK3 in neutrophil differentiation and survival of APL cells by Elena A. Federzoni, Peter J. M. Valk, Bruce E. Torbett, Torsten Haferlach, Bob Löwenberg, Martin F. Fey, and Mario P. Tschan Blood Volume 119(21):4963-4970 May 24, 2012 ©2012 by American Society of Hematology

Significantly reduced HK3 and PU Significantly reduced HK3 and PU.1 mRNA levels in primary AML patient samples and induction of HK3 expression during neutrophil differentiation of NB4 APL cells. Significantly reduced HK3 and PU.1 mRNA levels in primary AML patient samples and induction of HK3 expression during neutrophil differentiation of NB4 APL cells. (A) Primary AML blasts were isolated using a Ficoll gradient, total RNA was extracted, and HK3 (left panel) and PU.1 (right panel) mRNA levels were measured by real-time quantitative RT-PCR. Measured cycle threshold (Ct) values represent log2 expression levels. Values were normalized to the expression levels of the housekeeping genes HMBS and ABL1. ***P < .0001. ‡P < .05. (B) NB4 and NB4-R2 cells were differentiated with 1μM ATRA for 6 days. HK1, HK2, and HK3 mRNA were measured by real-time quantitative RT-PCR and given as n-fold changes compared with untreated SHCOO2 cells and normalized to the housekeeping gene HMBS. ***P < .0001. (C) PU.1 and HK3 protein expression was measured by Western blotting in NB4 and NB4-R2 treated as in panel B. GAPDH expression was used as loading control. (D) CEBPE mRNA expression was determined in NB4 cells treated as in panel B. ***P < .0001. (E) CD11b flow cytometric statistical analysis of NB4 or NB4-R2 cells treated as in panel B. *P < .01 (all P values Mann-Whitney U tests). Elena A. Federzoni et al. Blood 2012;119:4963-4970 ©2012 by American Society of Hematology

Transcriptional regulation of HK3 by PU Transcriptional regulation of HK3 by PU.1 in NB4 and HT93 APL cell lines. Transcriptional regulation of HK3 by PU.1 in NB4 and HT93 APL cell lines. NB4 and HT93 cells were stably transduced with nontargeting shRNA (SHC002) or shRNAs targeting PU.1 (shPU.1_256, shPU.1_928) and differentiated with 1μM ATRA for 6 days. HK3 and PU.1 mRNA expression as well as protein expression were measured in NB4 (A,C) or HT93 (B,D) cells by real-time quantitative RT-PCR and Western blotting. CEBPE mRNA expression was measured in NB4 (E) and HT93 (F) cells by real-time quantitative RT-PCR. CD11b flow cytometric analysis of NB4 (G) and HT93 (H) cells. ***P < .0001; **P < .01; *P < .05 (all P values Mann-Whitney U tests). Elena A. Federzoni et al. Blood 2012;119:4963-4970 ©2012 by American Society of Hematology

PU.1 and PML-RARA are direct regulators of the HK3 promoter. PU.1 and PML-RARA are direct regulators of the HK3 promoter. (A) Schematic representation of a 6.6-kb human HK3 promoter fragment. Lanes A, B, and C are 3 putative PU.1 binding sites (circles) found in the HK3 promoter as determined by MatInspector. (B) In vivo binding of PU.1 to the 3 binding sites was shown by ChIP in NB4 cells. ChIP was performed with antibodies against PU.1, PML, and RARA. Antibodies against acetyl-histone H3 and IgG served as positive and negative controls, respectively. As a negative control for the different pull-downs, absence of GAPDH amplification is shown. (C) HK3 promoter transactivation assays. H1299 cells were transiently transfected with 40 ng HK3 promoter reporter construct and PU.1 or PML-RARA expression vectors as indicated. Total amount of transfected DNA was adjusted with pcDNA3.1 empty vector as indicated. pRL-TK Renilla luciferase vector (40 ng) was cotransfected in each experiment as an internal control for transfection efficiency. After 24 hours, luciferase activity was measured. The promoter activity is shown as relative light units (RLU). Results are the mean ± SD of 5 independent experiments. ***P < .0001; **P < .001 (Mann-Whitney U tests). Elena A. Federzoni et al. Blood 2012;119:4963-4970 ©2012 by American Society of Hematology

Inhibition of HK3 by shRNA impairs neutrophil differentiation of APL cells. Inhibition of HK3 by shRNA impairs neutrophil differentiation of APL cells. NB4 cells were stably transduced with nontargeting shRNA (SHC002) or shRNAs targeting HK3 (shHK3_560, shHK3_1420, shHK3_1748) and differentiated with 1μM ATRA for 6 days. HK3 (A) and CEBPE (B) mRNA levels were measured by real-time quantitative RT-PCR and are given as n-fold changes compared with untreated SHCOO2 cells and normalized to the housekeeping gene HMBS. HK3 protein expression was measured by Western blotting. GAPDH expression was used as loading control. (C-D) CD11b flow cytometric analysis of NB4 control (SHCOO2) and HK3 knockdown cells.***P < .0005; **P < .005 Elena A. Federzoni et al. Blood 2012;119:4963-4970 ©2012 by American Society of Hematology

Inhibition of HK3 decreases cell viability of ATRA-differentiated APL cells and renders APL cells more sensitive to anthracyclin therapy. Inhibition of HK3 decreases cell viability of ATRA-differentiated APL cells and renders APL cells more sensitive to anthracyclin therapy. (A) NB4 control (SHCOO2) or HK3 knockdown (shHK3_560, shHK3_1420, shHK3_1748) APL cells were grown in the presence or absence of 1μM ATRA for 4 or 6 days. Cell viability was measured by an Alamar Blue assay in 3 independent experiments. Cell viability is shown as percent change compared with untreated SHCOO2 control cells. **P < .005, shHK3 vs SHC002 control (Mann-Whitney U test). (B) NB4 control (SHC002) or HK3 knockdown (shHK3_560, shHK3_1420, shHK3_1748) APL cells were incubated with 0.01μM idarubicin for 48 hours (left panel) or 0.03μM doxorubicin for 72 hours (right panel), and cell viability was measured as in panel A. **P < .01; *P < .05 (Mann-Whitney U tests). (C) Caspase activation of NB4 cells treated with 0.1μM idarubicin for 24 hours (left panel) or 0.1μM doxorubicin for 48 hours (right panel) was measured by a luminescence assay. ***P < .0001 (Mann-Whitney U test). Elena A. Federzoni et al. Blood 2012;119:4963-4970 ©2012 by American Society of Hematology