RUNX1 regulates phosphoinositide 3-kinase/AKT pathway: role in chemotherapy sensitivity in acute megakaryocytic leukemia by Holly Edwards, Chengzhi Xie, Katherine M. LaFiura, Alan A. Dombkowski, Steven A. Buck, Julie L. Boerner, Jeffrey W. Taub, Larry H. Matherly, and Yubin Ge Blood Volume 114(13):2744-2752 September 24, 2009 ©2009 by American Society of Hematology

Real-time RT-PCR quantification of RUNX1 transcripts in megakaryoblasts from children with and children without DS newly diagnosed with AMkL. Real-time RT-PCR quantification of RUNX1 transcripts in megakaryoblasts from children with and children without DS newly diagnosed with AMkL. Transcript levels for RUNX1a, RUNX1b/1c, and RUNX1c were measured by real-time RT-PCR and normalized to 18S rRNA levels. RUNX1b transcript levels were calculated by subtracting RUNX1c transcript levels from the total transcript levels for RUNX1b/1c. The horizontal lines indicate median RUNX1 transcript levels in each group of patient samples. The P value was determined by the nonparametric Mann-Whitney U test. Holly Edwards et al. Blood 2009;114:2744-2752 ©2009 by American Society of Hematology

shRNA knockdown of RUNX1 in Meg-01 cells resulted in increased sensitivity to ara-C. shRNA knockdown of RUNX1 in Meg-01 cells resulted in increased sensitivity to ara-C. (A-B) Meg-01 cells were infected by RUNX1 shRNA lentivirus clones. After selection with puromycin, infected Meg-01 cells were plated in soft agar. Colonies were isolated, expanded, and tested for RUNX1 expression by real-time RT-PCR (A) and Western blotting (B). Two colonies (designated RUNX1 1-1 and RUNX1 1-2) with decreased RUNX1 gene expression were selected as candidates for further study. A pool of cells from the negative control transduction was used as the control (designated negative). (C) The RUNX1 1-1, RUNX1 1-2, and negative cells were cultured in complete medium with dialyzed FBS in 96-well plates at a density of 4 × 104 cells/mL. Cells were cultured continuously with a range of concentrations of ara-C at 37°C, and cell numbers were determined with the Cell Titer–blue reagent and a fluorescence microplate reader. The data represent mean values ± SEs from at least 3 independent experiments. Holly Edwards et al. Blood 2009;114:2744-2752 ©2009 by American Society of Hematology

Overlapping genes between the oligonucleotide microarray dataset derived from Meg-01 shRNA stable clones and the Affymetrix microarray dataset derived from primary DS and non-DS AMkL patient blasts. Overlapping genes between the oligonucleotide microarray dataset derived from Meg-01 shRNA stable clones and the Affymetrix microarray dataset derived from primary DS and non-DS AMkL patient blasts. (A) Differentially expressed genes identified by oligonucleotide microarray from Meg-01–negative and RUNX1 1-1 cells (left circle) were cross referenced, by comparing GenBank accession numbers, with genes differentially expressed between primary DS and non-DS AMkL patient specimens identified by Affymetrix microarray (right circle) in our previous study.27 A total of 351 probes (supplemental Table 7) were identified in both microarray datasets (overlapping area), including PIK3CD. (B) Transcript levels for PIK3CD were quantitated by real-time RT-PCR in the Meg-01 shRNA stable clones to validate the oligonucleotide microarray data. Real-time PCR results were expressed as mean values from 3 independent experiments with the same cDNA preparation and normalized to 18S rRNA. **Statistically significant differences (P < .005). Error bars indicate SEs. (C-D) PIK3CD transcript levels in primary DS and non-DS AMkL blasts were quantitated by real-time RT-PCR. Median PIK3CD transcript levels were compared between the 2 patient groups with the use of the nonparametric Mann-Whitney U test (C), and their relation to RUNX1b transcript levels was determined by the nonparametric Spearman rank correlation coefficient (D). Holly Edwards et al. Blood 2009;114:2744-2752 ©2009 by American Society of Hematology

PIK3CD is a bona fide RUNX1 target gene in AMkL. PIK3CD is a bona fide RUNX1 target gene in AMkL. (A-C) In vivo binding of RUNX1 to the putative RUNX1 binding sites located in the upstream region of the PIK3CD gene (A) in Meg-01 cells was determined by ChIP assays with the use of regular PCR (B) and real-time PCR (C), as described in “Methods.” (D-E) KA1b cells were transfected with pGL3Basic-PIK3CDpro along with pRLSV40 by electroporation. The transfected cells were split into 2 equal aliquots with 1 induced by 2 μg/mL Dox. Cells were harvested 24 hours after transfection, and induction of RUNX1b was determined by Western blotting (D) and luciferase activities were assayed (E) as described in “Methods.” **Statistically significant differences (P < .005). (C and E) Error bars indicate SEs. Holly Edwards et al. Blood 2009;114:2744-2752 ©2009 by American Society of Hematology

Functional impact of RUNX1 on PI3-kinase/Akt signaling and cell apoptosis in Meg-01 cells. Functional impact of RUNX1 on PI3-kinase/Akt signaling and cell apoptosis in Meg-01 cells. (A) The RUNX1 1-1, RUNX1 1-2, and negative stable clones were lysed in PBS buffer containing both protease and phosphatase inhibitors. Soluble proteins were analyzed by Western blots with anti-p110δ, anti–ph-Akt, and anti-Akt antibodies. Intensity of each band was quantified with the use of the Odyssey software. Protein levels for p110δ and Ph-Akt in RUNX1 1-1 and RUNX1 1-2 cells are presented relative to that in negative cells after normalization to levels of total Akt. (B) Baseline apoptosis in the RUNX1 1-1, RUNX1 1-2, and negative stable clones was determined by flow cytometry with annexin V-fluorescein isothiocyanate/propidium iodide staining. **Statistically significant differences (P < .005). Error bars indicate SEs. Holly Edwards et al. Blood 2009;114:2744-2752 ©2009 by American Society of Hematology

Synergistic antileukemia activity between the PI3-kinase inhibitor, LY294002, and ara-C in Meg-01 cells and primary blasts from children without DS with AMkL. Synergistic antileukemia activity between the PI3-kinase inhibitor, LY294002, and ara-C in Meg-01 cells and primary blasts from children without DS with AMkL. (A) Meg-01 cells were harvested and lysed after incubation with a range of concentrations of the nonselective PI3-kinase inhibitor, LY294002, for 48 hours. Soluble proteins were subjected to Western blots probed with anti-Akt and anti–ph-Akt antibodies. (B) Meg-01 cells were continuously cultured in 96-well plates for 96 hours in dialyzed FBS in the presence of various concentrations of LY294002. Cell numbers were determined by the Cell Titer–blue reagents, as described in “Methods.” (C) Ara-C IC50 values were determined in Meg-01 cells in the presence of different concentrations of LY294002. *Statistically significant differences (P < .05); **statistically significant differences (P < .005). (B-C) Error bars indicate SEs. (D) Standard isobologram analysis of Meg-01 cell growth inhibition by LY294002 and ara-C in combination. The IC50 values of each drug are plotted; the solid line represents the additive effect, whereas the points represent the concentrations of each drug resulting in 50% growth inhibition. (E-F) Ara-C IC50 values were determined in the presence of a range of concentrations of LY294002 in 2 primary AMkL blasts from 2 children newly diagnosed with de novo AMkL by MTT assay as described in “Methods.” Holly Edwards et al. Blood 2009;114:2744-2752 ©2009 by American Society of Hematology