STAT3 phosphorylation is mediated by the tyrosine kinase JAK2 in NSCLC.

<p><b>A</b>, The indicated NSCLC lines were plated at a fixed density and allowed to adhere overnight. Cells were then treated for 16 hours with DMSO (con) or a 2-fold escalating dose of the JAK1/2 inhibitor ruxolitinib ranging from 0.25 to 4.0 µM (left-right). Protein lysates were harvested and the phosphorylation status of STAT3 (pSTAT3^Y705), AKT (pAKT^S473), ERK (pERK^T202/Y204), and S6 (pS6^S235/S236) was evaluated by immunoblot. Equal amounts of protein (20 µg) were added for each sample. <b>B</b>, The same NSCLC lines were plated as in A, but were treated with a fixed dose of ruxolitinib (1 µM) for the indicated time. Phospho-protein status was evaluated in 20 µg of protein per sample as in A. <b>C</b>, NCI-H1703 cells were infected with lentiviral vectors containing a control shRNA (NT, non-targeting) or one of two shRNAs targeted to human JAK2 (sh1 or sh2). Protein lysates were harvested and evaluated for JAK2 expression and STAT3 phosphorylation by immunoblot. Blots for total STAT3 and tubulin were included to demonstrate equal loading. <b>D</b>, Representative images of immunohistochemical stains for total JAK2, total STAT3 and phosphorylated STAT3 (pSTAT3^Y705) performed on a tissue microarray containing 245 pathologically verified human NSCLC samples. Images were captured from staining performed serial sections of the same tumor samples. <b>E</b>, Summary of staining results for JAK2, STAT3 and pSTAT3^Y705 on the NSCLC tissue microarray (<i>n</i> = 245). Note that all but one of the samples with positive nuclear pSTAT3^Y705 staining (52/53, 98.1%) were also positive for JAK2.</p

A diagrammatic representation of <i>sample class</i> enrichments for selected genes.

<p>Samples classified as M or U by the discretization approach are indicated by green and blue bars respectively. The genomic context of samples in each group are shown as white, black or red bars. A representative locus is shown for p16 (A), DLC1 (B), IGF1R (C) and IL17RB (D).</p

STAT3 phosphorylation is mediated by IL-6 family cytokines in NSCLC cell lines.

<p><b>A</b>, The indicated NSCLC lines were plated at fixed densities and allowed to adhere overnight in serum-containing media. Cells were then treated for 24 hours in serum-free media containing anti-gp130 neutralizing antibody or a control mouse IgG (2.0 µg/mL each). Protein lysates were harvested and the phosphorylation status of STAT3 (pSTAT3^Y705), AKT (pAKT^S473), ERK (pERK^T202/Y204), and S6 (pS6^S235/S236) was evaluated by immunoblot. Equal amounts of protein (30 µg) were added for each sample. <b>B</b>, NSCLC lines were plated at fixed densities and allowed to adhere overnight in serum-containing media. Cells were then serum-starved, and conditioned media was collected at 48 hours. Secreted interleukin-6 (IL-6) levels were measured from the indicated cell lines using a bead-based immunoassay platform. Values shown are averages of duplicate measurements for each sample.</p

Methods used to identify genes with methylation-expression correlations.

<p>(A) Pearson correlation was used to measure linear relationships between DNA methylation and gene expression levels for 1505 CpG probes represented on the GoldenGate Methylation BeadArray. The panels represent examples of a gene with high (left) and low (right) Pearson correlation coefficients when analyzing DNA methylation levels (x axis) against gene expression levels (y axis). (B) A discretization approach was used to classify samples into <i>methylated</i> (M) or <i>unmethylated</i> (U) groups based on the mean (<i>μ</i>) methylation value and standard deviation (<i>σ</i>) of a given probe. Statistically significant gene expression differences between M and U groups indicated a methylation-expression correlation for the gene in question.</p

Targeted kinase inhibitors fail to downregulate STAT3 phosphorylation in NSCLC cell lines with diverse driver mutations.

<p><b>A</b>, KRAS mutant NSCLC cell lines (A549, NCI-H358 and NCI-H460) were treated for one hour with DMSO (con) or a 2-fold escalating dose of the MEK1/2 inhibitor U0126 ranging from 0.625 to 5.0 µM (left-right). The phosphorylation status of STAT3 (pSTAT3^Y705), AKT (pAKT^S473), ERK (pERK^T202/Y204), and S6 (pS6^S235/S236) was evaluated by immunoblot. Equal amounts of protein (20 µg) were added for each sample. <b>B</b>, PDGFRA amplified/mutant NSCLC cells (NCI-H1703) were treated for one hour with DMSO (con) or a 2-fold escalating dose of the PDGFRA inhibitor sunitinib ranging from 0.625 to 2.5 µM (left-right). Phospho-protein status was evaluated in 20 µg of protein per sample as in A. <b>C</b>, MET amplified NSCLC cells (NCI-H1993) were treated for one hour with DMSO (con) or a 2-fold escalating dose of the MET/ALK inhibitor crizotinib ranging from 0.25 to 1.0 µM (left-right). Phospho-protein status was evaluated in 20 µg of protein per sample as in A. <b>D</b>, The indicated NSCLC lines were plated at fixed densities and allowed to adhere for 24 hours. Cells were then treated for 18 hours in normal media with (+) or without (−) 10% fetal bovine serum (FBS). Phospho-protein status was evaluated in 20 µg of protein per sample as in A. Even sample loading is indicated by replicate immunoblot for beta-tubulin.</p

Top 40 methylation loci with statistically significant methylation-expression correlations using the <i>discretization approach</i>.

<p>Additionally, loci in common with the Pearson correlation method are shown in parentheses.</p>*<p>(−) indicates a negative methylation-expression correlation, where high methylation correlates to lower expression or vice versa. (+) indicates a positive correlation where high methylation correlates to increased expression or vice versa.</p>†<p>P values were obtained after 10,000 random permutations of each class label.</p>‡<p>Gene was selected for further validation.</p>§<p>The methylation level of the probe FRZB_P406_F has a positive correlation with gene expression by both Pearson correlation and the discretization method, while the other probe FRZB_E186_R has a negative methylation-expression correlation by both methods. Only the FRZB_P406_F probe is shown for the discretization method due to its statistical significance.</p

Comparison of Pearson and discretization approaches used to identify methylation-expression correlations.

<p>The Venn Diagram displays the total number of overlapping loci with positive/negative methylation-expression correlations identified by computing either a Pearson correlation or applying the discretization method.</p

JAK2 inhibition with ruxolitinib inhibits NSCLC cell growth in soft agar and in xenograft assays.

<p><b>A–B</b>, NSCLC cells were plated into a soft-agar suspension (5,000 cells/35 mm well) and overlaid with normal media containing 10% FBS +/− 2.0 µM ruxolitinib. After one month, agar plugs were fixed and stained with crystal violet to identify individual colonies. Digital images of each well were captured and analyzed for colony number and size. Average colony formation efficiency ([# colonies/# plated cells] ×100%, <b>A</b>) and mean colony size (area in µm², <b>B</b>) for triplicate experiments are shown. Error bars indicate the standard deviation for triplicate wells. (*, <i>p-value</i><0.05). <b>C</b>, HCC827 cells were xenografted into the flanks of nude mice. When tumors reached a volume of 100 mm³, mice were separated into two treatment arms (<i>n</i> = 6, each) and dosed daily by oral gavage with vehicle or the JAK2 inhibitor ruxolitinib (50 mg/kg). Tumor size was evaluated every other day. Animals were sacrificed when tumors reached 500 mm³ or after one month of treatment. Error bars indicate the standard error of tumor volume measurements. (*, <i>p-value</i><0.05).</p

Relationship to CpG islands and TSS for genes with methylation-expression correlations identified by the <i>discretization approach</i>.

*<p>Hypergeometric <i>P</i> values are given.</p

Summary of assay details for CpG sites interrogated by bisulfite pyrosequencing.

<p>Summary of assay details for CpG sites interrogated by bisulfite pyrosequencing.</p