Combination therapy with GDC0941 and AS-605 demonstrates <i>in vitro</i> efficacy.

<p><b>A.</b> Table of IC50 values for GDC-0941 and AS-605 showing effect on different PI3K isoforms. Source: Selleckchem.com <b>B.</b> Cells were seeded in the presence of each inhibitor or the combination. Bar graphs show the effect of combining GDC0941 and AS-605 inhibitors on growth of 1156 and 1713 T-ALL cell lines. Cell counts of the DMSO-treated control were set at 100% and the effect of each of treatment was calculated as a proportion of that. <b>C.</b> Histograms showing baseline phospho-Akt (S473) levels in human T-ALL cell lines (red line). Grey-filled histograms depict unstained controls. <b>D.</b> Bar graphs showing the effect of combining GDC0941 and AS-605 inhibitors on growth of human T-ALL cell lines. As in (B) cell counts of the DMSO treated control were set at 100% and the effect of each of treatment was calculated as a proportion of that.</p

PI3K-Akt signals in T cell leukemia are affected by doxorubicin treatment.

<p><b>A-C.</b> Western blot analysis of basal and cytokine-induced p-Akt (S473) in mouse (A-B) and human T-ALL cell lines (C). 1156, 1713, T-ALL C6 and C7 are mouse T-ALL cell lines with leukemia virus insertions in <i>RasGRP1</i> causing RasGRP1 overexpression (A). T-ALL 3, T-ALL 9 and T-ALL 15 are mouse T-ALL cell lines which harbor a KRas^G12D mutation. Representative blots of three or more (A & B) and two (C) independent experiments. Blotting for total Akt or αTubulin serves to demonstrate equal loading.</p

Combining GDC0941 and AS-605 inhibitors does not show <i>in vivo</i> efficacy.

<p><b>A.</b> Diagram showing preclinical trial set-up. Four days after transplant of primary JW-81 T-ALL cells, mice were randomized into four treatment arms: vehicle control, GDC0941, AS-605, or GDC0941 + AS-605. Three mice were enrolled on the vehicle control arm and five mice were enrolled on each treatment arm. <b>B.</b> Kaplan-Meier graph showing percentage of mice surviving treatment with PI3K inhibitors as a function of time. Curves are not significantly different (Mantel-Cox test). Median survival was 14 days post-transplant for vehicle control, AS-605 and GDC0941+AS-605 and 15 days for GDC0941-treated mice. <b>C.</b> Southern blot of analysis of MOL4070 integration sites in bone marrow from 13 T-ALL samples from the four preclinical trial arms. Note that no new bands are present in samples harvested from treated mice. Each lane represents bone marrow from one animal. <b>D.</b> Western blot analysis of bone marrow from 13 T-ALL samples from the four preclinical trial arms with each lanes representing one animal. Dots on the left indicate blot identities. Arrows on the right indicate bands that represent specific phospho-signals.</p

AS-6052425, a PI3Kγ inhibitor, effectively blocks phospho-Akt expression in T-ALL cell lines.

<p><b>A.</b> IC50 values (μM) of compounds used in this study for major targets. Source: Selleckchem.com <b>B-E</b>. Heatmaps of phosho-Akt (S473) signals in T-ALL cell lines (columns) stimulated with cytokines for 0, 2, 5 or 10 minutes (rows). Cells were treated with indicated drugs (at 10 μM unless otherwise indicated) or vehicle for 30 minutes prior to stimulation. Representative heatmaps from at least three experiments. <b>F</b>. Western blot analysis of phospho-Akt (T308 and S473) signals in 1713, C6 and C7 T-ALL cell lines treated with 10 μM GDC0941 (pan-PI3K inhibitor), 10 μM AS-605 (PI3K γ specific inhibitor) or vehicle (DMSO) and stimulated with cytokines IL-2/7/9. Representative blot from at least two experiments. Blotting for total Akt serves to demonstrate equal loading.</p

Pan- and γ-specific PI3K inhibitors have limited cytostatic effects on T-ALL cells.

<p><b>A.</b> (Left) Western blot analysis of phospho Akt (S473) and phospho pS6 signals in C6 T-ALL cell line treated with 10 μM PIK90 (pan-PI3K inhibitor), 10 μM AS-605 (PI3K γ-specific inhibitor) or vehicle control (DMSO) for indicated times. (Right) Quantification of the Western blot shown on the left. The intensity of pAkt signal was normalized against total Akt and DMSO control was set to 100% for each time point. <b>B-C</b> Representative FACS dot plots showing gating of apoptotic cells that are double positive for cleaved caspase-3 and cleaved PARP. Left bar graph shows induction of apoptosis by AS-605 (B) or GDC0941 (C) after 8 hours of treatment. Graph on the right shows the percentage of viable cells after 68 hours of inhibitor treatment. Data were normalized to DMSO control, which was set as 100%. One representative example out of three independent experiments is shown. Error bars are SD of duplicates. <b>D.</b> Histogram showing CFSE dilution of 1156 T-ALL cell line treated with AS-605, GDC0941 or vehicle control 44 hours after drugs were added. The effect of drugs on proliferation was measured by calculating proliferation index (ratio of geometric mean of DMSO over drug treated samples). Shown is mean of two or three experiments ±SD.</p

Supplementary Material, DS_DISC774248 – High-Throughput Flow Cytometry Identifies Small-Molecule Inhibitors for Drug Repurposing in T-ALL

<p>Supplementary Material, DS_DISC774248 for High-Throughput Flow Cytometry Identifies Small-Molecule Inhibitors for Drug Repurposing in T-ALL by Dominique R. Perez, Christian K. Nick, Anna Waller, Cristina Delgado-Martin, Travis Woods, Nitesh D. Sharma, Michelle L. Hermiston, Mignon L. Loh, Stephen P. Hunger, Stuart S. Winter, Alexandre Chigaev, Bruce Edwards, Larry A. Sklar and Ksenia Matlawska-Wasowska in SLAS Discovery</p