Evaluation of selumetinib and trametinib Mek1/2 inhibitors as mono-treatment on BCP-ALL cell viability in the presence and absence of stroma.

<p>The indicated ALLs were treated with different μM concentrations of selumetinib or trametinib for 72 hours in the presence (A) or absence (B) of irradiated OP9 stromal support. Note: “0” values were determined on one sample set but are shown twice, for selumetinib and trametinib, for clarity. (C) 72-hour treatment of primary LAX56 with selumetinib as indicated. Viability (left panels) and cell counts (right panels) were determined by Trypan Blue exclusion. Error bars, SD of values measured in triplicate wells. One of two experiments for US7 and TXL2 with similar results. *p<0.05 **p<0.01, one-way ANOVA.</p

Diagnosis ALL samples differ in ability to activate Erk1/2 upon serum stimulation.

<p>Diagnosis sample t[(1;4)(p22;q31)](A, D); LAX40 [t(8;14)(q24;q32)] (B, E); or LAX39 (TEL-AML1) (C, F) were assayed for serum-stimulated generation of pErk1/2 (CST antibodies) by phospho-flow (A-C) or Western blot (D-F) in the presence or absence of selumetinib. (A) Cells were directly stimulated with 20% FBS in αMEM (day 1, left panel) or placed overnight in X-Vivo15 prior to stimulation with 20% FBS and stroma (day 2, right panel) for the indicated period of time in the presence of solvent DMSO or 10 μM selumetinib. (B, C) ALL cells in X-Vivo15 medium for 3 hrs were stimulated with 20% FBS in αMEM in the presence of 10 μM selumetinib or DMSO as indicated. MFI, mean fluorescent intensity. (D, E, F) Samples harvested from the same cultures used for phospho-flow. (D) Western blot on day 2 samples. (E, F) serum αMEM; 20% FBS in αMEM. Three different diagnosis samples, each analyzed once.</p

Western blot analysis shows pErk reduction through selumetinib treatment and by removal of stromal support.

<p>ICNO6 and TXL2 ALL cells were cultured for 4 hrs in αMEM + 20% FBS on OP9 stroma or in αMEM + 1% BSA without OP9 stroma, and then treated in the same media for an additional 4 hours with 10 μM selumetinib. Western blots were incubated with the antibodies indicated in the panel. Gapdh, loading control. The membrane was sequentially stripped and re-probed with antibodies.</p

Viability and proliferation of ALL cells treated with trametinib and CAL101.

<p>US7 or TXL2 cells (10⁶ cells) as indicated were treated for 72 hours with 10 μM trametinib, 10 μM CAL101, or a combination of the two. Viability and cell counts were determined by Trypan blue exclusion. Cells were either exposed to drug while on stroma (A) or without stroma (B) in complete medium with 20% FBS. Error bars, mean ±SEM of triplicate values. *p<0.05; **p<0.01, one-way ANOVA.</p

Phospho-flow detects increased pErk1/2 in US7R relapse sample with an activating K-Ras mutation compared to matched diagnostic US7 sample lacking the mutation.

<p>Cells starved overnight in X-Vivo15 medium were stimulated for 4 hours with αMEM + 20% FBS and OP9 stroma in the presence of solvent DMSO or 10 μM selumetinib. Controls (t 0 samples) continued to be starved in αMEM with 1% BSA in the absence of stroma. US7 and US7R cells were assayed for serum-stimulated generation of pErk1/2 by phospho-flow using pErk1/2 (CST, left panel) or pMek (BD, right panel) antibodies. Error bars, mean +/- SEM of three independent experiments. *p<0.05 **p<0.01. Student's t-test.</p

Phospho-flow cytometry detects Mek pathway activation and inhibition.

<p>(A) PBMC from normal donors were stimulated with 40 nM PMA for 15 minutes, then analyzed by phospho-flow. Left, pMek, right pErk. Error bars, mean ± SD of 3 independent samples. *p<0.05, Student's t-test. (B, C) Imaging cytometry analysis on PBMC not stimulated (B top) or stimulated with 40 nM PMA (B bottom) or LAX56 cells treated with DMSO (C, top) or 10 μM selumetinib (C, bottom) for 10 minutes. (B, C) left, representative images; right, summary plot. Analysis of 3500 to 20,000 events per condition; numbers in the brightfield images represent the number designation of the individual event shown. (D) Histograms of pErk1/2 phospho-flow analysis on TXL2 ALL cells cultured without stroma or FBS (left panel), or on OP9 stroma + 20% FBS (right panel), for 24 hours. Cells were treated for 4 hours with solvent DMSO (red), with 100 μM sodium pervanadate (purple), with 10 μM selumetinib (blue), or with 100 μM sodium pervanadate, plus 10 μM selumetinib (green) as indicated. Grey histograms, unstained controls; black line, secondary antibody only. Single analysis.</p

Mek pathway inhibition in combination-treated BCP-ALL cells by phospho-flow and Western blot.

<p>(A) Phospho-flow on US7 cells using pErk1/2 (CST) or pMek (BD) antibodies in the left and right panels respectively, as indicated, in the presence or absence of trametinib, CAL101 or both. All samples were starved overnight in X-Vivo15 medium (t 0 samples), then treated from t 0 onward for 4 hours with αMEM + 20% FBS and OP9 stroma (‘stimulated’), or with αMEM + 1% BSA in the absence of stroma (‘starved’). At t 0 solvent DMSO or 10 μM of the indicated drugs was also added to the samples. Error bars, mean +/- SEM of three independent experiments. *p<0.05 **p<0.01, Student's t-test. (B) Western blot analysis (single analysis) of US7 cells grown for 24 hours in αMEM + 20% FBS and OP9 stroma (‘OP9’ samples, left panel), or in αMEM + 1% BSA in the absence of stroma (‘no OP9’ samples, right panel). Cells were then additionally treated for 4 hours with solvent DMSO (control) or 10 μM of the indicated drugs. Gapdh, loading control. Western blot membrane was sequentially stripped and reprobed with antibodies.</p

Lymph node and splenic CD44⁺ T cells after arterial injury in WT mice.

<p>Representative scatter plots of lymph node (LN) and spleen cells collected at various times after injury and characterized using CD44 gated on CD4 (A) or CD8b (B). Cells were collected from uninjured (UI) mice, or 7 days (D7) and 21 days (D21) after arterial injury. Sham mice correspond to 7 days after sham surgery. Percentage of cells is indicated on the top right corner of each graph.</p

T cells in the injured arterial wall.

<p>Representative sections of 21-day injured carotid arteries stained for CD4 (A) or CD8b (B) identify their localization (arrows) in the arterial wall. Omission of primary antibody (C) was used as control for staining. N = 4 each; bar = 10 microns.</p

Splenic CD8b⁺ T cells after arterial injury in WT mice.

<p>Representative scatter graphs of CD8b-gated CD25⁺ (A, top panel) and CD28⁺ (A, bottom panel) spleen T cells. Representative histogram of CD28 expression on CD8b-gated cells (B). Geometric mean fluorescence intensity (MFI) of CD28 on CD8b-gated cells (C; N = 3–4 each time point).</p