Knockdown of p11 reduced ALL cell binding to osteoblasts.

<p><b>A)</b> Nalm6 cells were transfected with either scrambled or p11-specific shRNA (sequence 1 or sequence 2). Forty-eight hours post transfection, cells were plated on poly-lysine coated slides, fixed, stained with anti-p11 antibody followed by Alexa Flour 488-conjugated anti-mouse secondary antibody, and visualized by confocal microscopy. Scale bar = 10 μm. Representative images in gray scale are shown. <b>B)</b> The mean fluorescence intensity corresponding to p11 protein levels was determined from 100 cells per condition. The error bars denote SD of the Mean (*P < 0.01). <b>C)</b> Transfected cells were subjected to adhesion assay as described in Materials and Methods. Error bars denote SD of the Mean from two independent experiments in quadruplicates. Asterisks indicate statistical significance (*P < 0.001).</p

ANX2/p11 interaction mediates binding of ALL cells to osteoblasts.

<p><b>A)</b> Cell adhesion assay showing the percentage of RS4;11 cells bound to Saos-2 monolayer with respect to the input, which was considered 100%. Error bars denote SD of the Mean from three independent experiments. Asterisk indicates statistical significance, P < 0.01. <b>B)</b> Immunoprecipitates using control IgG or anti-p11 antibody from lysates of Nalm6 cells treated with or without ANX2T inhibitor (50 μM) were blotted using ANX2 or p11 antibodies. Cell lysate corresponding to 10% of the total protein input in the co-immunoprecipitation assay was loaded in the lane denoted as “Lysate”. <b>C)</b> Graph shows the percentage of cells that bound to Saos-2 monolayer in the presence or absence of ANX2T inhibitor with respect to the input. Error bars indicate SD of the Mean from three to four independent experiments. Note the dose dependent decrease in the percentage of bound Nalm6 and RS4;11 cells post treatment with ANX2T inhibitor (*P < 0.05, **P < 0.01).</p

p11 is upregulated at mRNA and protein levels and is localized to the cell surface in ALL cells.

<p><b>A)</b> Analysis of microarray data from GSE7440 showing fold-increase in transcript levels of ANX2 and p11. CCR = patients under continuous complete remission for more than four years (n = 28). Relapse = patients who relapsed within three years of initial diagnosis (n = 31). Error bars denote Standard Deviation (SD) of the Mean. Asterisk indicates statistical significance with a confidence limit of 95%. <b>B)</b> Quantitative RT-PCR data showing mRNA levels of ANX2 and p11 in pediatric normal immortalized B-cells (AG09390 and AG15007) and pediatric (Nalm6, REH) or adult (RS4;11) pre B-ALL cell lines. Error bars denote SD of the Mean from four independent experiments. Asterisks indicate statistical significance with a confidence limit of 95%. <b>C)</b> Immunoblots showing ANX2 and p11 levels in the indicated cell lines. GAPDH was used as a loading control. Representative blots from three independent experiments are shown. <b>D)</b> Representative immunoblots showing ANX2 and p11 levels in lysates from mononuclear cells isolated from healthy volunteers (Normal 1 and 2), or from leukemic cells from pediatric patients. Samples were collected using IRB approved protocols. <b>E)</b> Flow cytometry profiles of fixed, non-permeabilized RS4;11 cells incubated with (red trace) or without (blue trace) addition of the indicated primary antibodies. <b>F)</b> Mean fluorescence intensity was plotted from three independent experiments. Error bars denote SD of the Mean. Asterisks indicate statistical significance (P < 0.01).</p

ANX2T inhibitor sensitizes primary ALL cells co-cultured with osteoblasts to dexamethasone and vincristine.

<p><b>A)</b> Graph shows the percentage of NTPL-20 cells that bound to Saos-2 monolayer in the presence or absence of ANX2T inhibitor or anti-ANX2 antibody with respect to the input. Error bars indicate SD of the Mean from three independent experiments. Asterisks denote P < 0.01. <b>B)</b> Primary ALL cells (NTPL-20) were either plated on tissue culture plastic or Saos-2 monolayers and treated with 1 μM dexamethasone or 10 nM vincristine. Twenty-four hours post incubation, leukemic cells were collected by vigorous pipetting and stained with propidium iodide. The percentage of viable cells in the population was determined by flow cytometry based on exclusion of propidium iodide. Graph represents the percentage of viable cells in each sample normalized to the control (cells plated on osteoblasts in the presence of DMSO considered as 100%). Asterisks indicate statistical significance (*P < 0.05, **P < 0.01). <b>C</b>) Representative images of histological sections of femur from a mouse transplanted with NTPL-20 cells. Dual immunohistochemistry staining was performed showing osteocalcin (osteoblast specific marker, in brown) and CD45 (marker for ALL cells, in red). The black square in the upper panel marks the region of the slide, which is magnified in the lower panel. Scale bars = 100 μm. Similar data was obtained in three different mice.</p

Disruption of interaction between ANX2 and p11 suppresses short-term homing and engraftment of primary ALL cells in immune-compromised mice.

<p><b>A)</b> NSG-B2m mice were injected with either anti-ANX2 antibody or an isotype-matched control antibody (IgG) before transplantation of 10 million NTPL-20 cells. Twenty-four hours post cell injection, mice were sacrificed and peripheral blood, spleen, liver, and femurs were harvested. Femurs were flushed to collect bone marrow. The percentage of human cells was detected by flow cytometry. The error bars denote SE of the Mean (n = 6 each). Asterisks indicate statistical significance with P < 0.05. <b>B)</b> NSG-B2m mice were injected with either anti-p11 antibody or an isotype-matched control antibody (IgG) before transplantation of 10 million NTPL-20 cells. The percentage of human cells in mouse organs was detected by flow cytometry twenty four hours post transplantation as described in A. The error bars denote SE of the Mean (n = 5 each). Asterisks indicate statistical significance with P < 0.05. <b>C</b>) Mice were pre-treated with either ANX2T inhibitor or vehicle before NTPL-20 cell transplantation. The graph shows the percentage of human cells in indicated organs determined twenty-four hours post cell injection. The error bars represent SE of the mean (n = 5). Asterisk denotes P < 0.05. <b>D</b>) Mice were pre-treated with either ANX2T inhibitor or vehicle before NTPL-90 cell transplantation. The graph shows the percentage of human cells in indicated organs determined twenty-four hours post cell injection. The error bars represent SE of the mean (n = 5). Asterisk denotes p < 0.05. <b>E)</b> The graph shows the increase in the percentage of human cells in mouse blood over time. Error bars denote SE of the mean from five mice each. *P < 0.05. <b>F)</b> Percentage of engraftment in bone marrow and spleen in mice at two weeks post transplantation with NTPL-20 cells is shown in the graph. Error bars represent SE of the mean from four mice each. *P < 0.01.</p

Vorinostat Enhances Cytotoxicity of SN-38 and Temozolomide in Ewing Sarcoma Cells and Activates STAT3/AKT/MAPK Pathways

<div><p>Histone deacetylase inhibitors (HDACi) have been evaluated in patients with Ewing sarcoma (EWS) but demonstrated limited activity. To better understand the potential for HDACi in EWS, we evaluated the combination of the HDACi vorinostat, with DNA damaging agents SN-38 (the active metabolite of irinotecan and topoisomerase 1 inhibitor) plus the alkylating agent temozolomide (ST). Drugs were evaluated in sequential and simultaneous combinations in two EWS cell lines. Results demonstrate that cell viability, DNA damage and reactive oxygen species (ROS) production are dependent on the sequence of drug administration. Enhanced cytotoxicity is exhibited <i>in vitro</i> in EWS cell lines treated with ST administered before vorinostat, which was modestly higher than concomitant treatment and superior to vorinostat administered before ST. Drug combinations downregulate cyclin D1 to induce G0/G1 arrest and promote apoptosis by cleavage of caspase-3 and PARP. When ST is administered before or concomitantly with vorinostat there is activation of STAT3, MAPK and the p53 pathway. In contrast, when vorinostat is administered before ST, there is DNA repair, increased AKT phosphorylation and reduced H2B acetylation. Inhibition of AKT using the small molecule inhibitor MK-2206 did not restore H2B acetylation. Combining ST with the dual ALK and IGF-1R inhibitor, AZD3463 simultaneously inhibited STAT3 and AKT to enhance the cytotoxic effects of ST and further reduce cell growth suggesting that STAT3 and AKT activation were in part mediated by ALK and IGF-1R signaling. In summary, potent antiproliferative and proapoptotic activity were demonstrated for ST induced DNA damage before or simultaneous with HDAC inhibition and cell death was mediated through the p53 pathway. These observations may aid in designing new protocols for treating pediatric patients with high-risk EWS.</p></div

STAT3, AKT and MAPK activation in EWS cell lines following exposure to ST and vorinostat.

<p><b>(A)</b> Immunoblot analysis of lysates of A4573 and TC32 cells following exposure to single (ST and V) and combination (ST/V, V/ST and STV) drug treatments, using antibodies against STAT3, p-STAT3 (Y705), AKT, p-AKT (S473), MAPK, p-MAPK (p42/44), histone 2B (H2B), and ac-H2B (K5). GAPDH was loading control. <b>(B)</b> HDAC activity was measured in A4573, and <b>(C)</b> TC32 cell lysates using the HDAC Activity Assay Kit (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142704#sec002" target="_blank">Methods</a>). Data are presented as mean absorbance (±SE), n = 3. Asterisks denote significant differences for drug combinations, **p< 0.01.</p

Summary of EWS cellular response to ST and vorinostat drug combinations.

<p>The figure depicts EWS cellular response following DNA damage (DNA double-strand breaks and ROS production) and HDAC inhibition. ST promotes DNA damage and vorinostat inhibits HDACs leading to activation of p53, inhibition of CD1, cleavage of caspase-3 and induction of apoptosis. A secondary drug response involves activation of STAT3, mediated by Src, and activation of AKT and MAPK mediated in part through ALK and IGF-1R. V/ST combination facilitates greater cell proliferation (Survival) and ST/V and STV combinations facilitate greater cell death (Apoptosis). Abbreviations: HDAC—Histone deacetylase; ROS—reactive oxygen species, CD1 –cyclin D1, STAT3 –signal transducer and activator of transcription 3; MAPK—mitogen-activated protein kinase, ALK—anaplastic lymphoma kinase; IGF-1R –insulin-like growth factor 1 receptor. Red arrows represent drug inhibition; Solid gray arrows represent constitutive signaling pathways; Solid blunt lines represent inhibition of signals; Dotted gray lines represent activation of signaling pathways in response to drug treatments.</p

STAT3, AKT and MAPK inhibition in EWS cell lines following exposure to ST and vorinostat, MK-2206 and AZD3463.

<p><b>(A)</b> Immunoblot analysis of lysates of A4573 and TC32 cells following exposure to media only (Control, C); ST/V and V/ST with (+) or without (-) 5.0 μM MK-2206 using antibodies against AKT, p-AKT (S473), HDAC1, histone 2B (H2B) and ac-H2B (K5). GAPDH was loading control. <b>(B)</b> Immunoblot analysis of A4573 and TC32 cells following exposure to single (ST and V) and combination (ST/V, V/ST and STV) drug treatments, using an antibody against MGMT. <b>(C)</b> A4573 cells and <b>(D)</b> TC32 cells were incubated with media only (Control, C); ST/V and V/ST with (+) or without (-) 20 nM AZD3463 and viable cells were measured at 48 h by the MTT assay. Data represents mean absorbance (±SE), n = 6. Asterisks denote significant differences between (+) versus (-) 20 nM AZD3463, **p< 0.01 <b>(E)</b> Immunoblot analysis of lysates of A4573 and TC32 cells following exposure to media only (Control, C); ST/V and V/ST with (+) or without (-) 20 nM AZD3463 using antibodies against ALK, IGF-1R, STAT3 (Y705), p-STAT3, AKT, p-AKT (S473), MAPK, p-MAPK (p42/44).</p

DNA damage and intracellular ROS generation in EWS cells following exposure to ST and vorinostat.

<p><b>(A)</b> Immunoblot analysis of γ-H2AX in lysates of A4573 and TC32 subjected to single agents (ST and V) and drug combination (ST/V, V/ST and STV) treatments. GAPDH was loading control. <b>(B)</b> Comet images illustrating DNA fragmentation in TC32 cells treated with single agents and drug combinations. Comet assays were performed in triplicate. <b>(C)</b> The tail intensity relative to the total cell DNA intensity is represented as the percent tail DNA for each treatment group. Data shown represent mean ± SE for each experimental group (n = 50 cells). <b>(D)</b> The tail lengths of comets were measured in individual cells and expressed relative to the control group. Data shown represent mean ± SE for each experimental group (n = 50 cells). Asterisks denote significant differences for drug combinations, **p< 0.01. Asterisks denote significant differences in drug combinations, **p< 0.01 <b>(E)</b> Intracellular ROS production in TC32 cells following single and combination treatments was determined using dichlorofluorescein acetate (DCFA) dye. ROS fluorescence was quenched by N-acetyl-cysteine (NAC). Emitted fluorescence (Em/Ex 490/530 nm) in treated cells was compared with untreated control cells. Measurements were taken for 2 independent experiments, and the data are mean ± SE. Asterisks denote significant differences for drug combinations, **p< 0.01.</p