Microarray and Proteomic Analyses of Myeloproliferative Neoplasms with a Highlight on the mTOR Signaling Pathway

The gene and protein expression profiles in myeloproliferative neoplasms (MPNs) may reveal gene and protein markers of a potential clinical relevance in diagnosis, treatment and prediction of response to therapy. Using cDNA microarray analysis of 25,100 unique genes, we studied the gene expression profile of CD34(+) cells and granulocytes obtained from peripheral blood of subjects with essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). The microarray analyses of the CD34(+) cells and granulocytes were performed from 20 de novo MPN subjects: JAK2 positive ET, PV, PMF subjects, and JAK2 negative ET/PMF subjects. The granulocytes for proteomic studies were pooled in 4 groups: PV with JAK2 mutant allele burden above 80%, ET with JAK2 mutation, PMF with JAK2 mutation and ET/PMF with no JAK2 mutation. The number of differentially regulated genes was about two fold larger in CD34(+) cells compared to granulocytes. Thirty-six genes (including RUNX1, TNFRSF19) were persistently highly expressed, while 42 genes (including FOXD4, PDE4A) were underexpressed both in CD34(+) cells and granulocytes. Using proteomic studies, significant up-regulation was observed for MAPK and PI3K/AKT signaling regulators that control myeloid cell apoptosis and proliferation: RAC2, MNDA, S100A8/9, CORO1A, and GNAI2. When the status of the mTOR signaling pathway related genes was analyzed, PI3K/AKT regulators were preferentially up-regulated in CD34(+) cells of MPNs, with down-regulated major components of the protein complex EIF4F. Molecular profiling of CD34(+) cells and granulocytes of MPN determined gene expression patterns beyond their recognized function in disease pathogenesis that included dominant up-regulation of PI3K/AKT signaling

Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.

Expression of genes linked to mTOR signaling pathway in CD34⁺ cells of MPN origin.

<p>A) Up-regulated genes vs. HuURNA in ET, PV, PMF and ET/PMF subjects without <i>JAK2V617F</i> mutation (Mut0); B) Unchanged gene expression vs. HuURNA in ET, PV, PMF and Mut0 subjects; C) Down-regulated genes vs. HuURNA in ET, PV, PMF and Mut0 subjects analyzed by microarray. Values are mean ± SEM (n = 4–9). *P < 0.05 compared among MPNs.</p

mTOR signaling pathway in CD34⁺ cells of MPN origin.

<p>(+p) phosphorylation, (-p) dephosphorylation; → stimulation, ┴ inhibition; white boxes represent down-regulated genes, gray boxes unchanged genes, black boxes up-regulated genes <i>vs</i>. HuURNA (corresponding to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135463#pone.0135463.g004" target="_blank">Fig 4</a>), while blue boxes represent none expressed or sporadically expressed genes in MPNs. GFR—growth factor receptors.</p

The statistically significant genes (p<0.01) among MPNs in CD34⁺ cells determined by microarray analysis.

<p>The negative values represent down-regulated genes, while positive values represent up-regulated genes compared to HuURNA. BG—Between groups significance with p<0.01, MD—Maximal mean difference of corresponding BG.</p><p>The statistically significant genes (p<0.01) among MPNs in CD34⁺ cells determined by microarray analysis.</p

Comparison of total gene expression in CD34⁺ cells among MPNs by Venn diagram after 50% filtration.

<p>A) Among <i>JAK2V617F</i>+ ET, PV and PMF; Among <i>JAK2V617F</i> negative ET/PMF (Mut0) and B) <i>JAK2V617F</i>+ ET and PV; C) <i>JAK2V617F</i>+ ET and PMF; D) <i>JAK2V617F</i>+ PV and PMF. Table represents the genes exclusively expressed in a single MPN disorder.</p

The common statistically significant genes in comparative proteomic (granulocytes) and microarray analyses (CD34⁺ cells).

<p>Scores—proteomic results (P) bolded (p<0.05) and non bolded (p<0.01); microarray results (M) with ratio > 1 (A1: p<0.05, A2: p<0.01) and ratio < 1 (B1: p<0.05, B2: p<0.01). PV/ET—polycythemia vera / <i>JAK2V617F</i>+ essential thrombocythemia ratio, PV/PMF—PV / <i>JAK2V617F</i>+ primary myelofibrosis ratio, PV / Mut0—PV / <i>JAK2V617F</i>- ET/PMF ratio.</p><p>The common statistically significant genes in comparative proteomic (granulocytes) and microarray analyses (CD34⁺ cells).</p

Comparison of total gene expression in granulocytes among MPNs by Venn diagram after 50% filtration.

<p>A) Among <i>JAK2V617F</i>+ ET, PV and PMF; Among <i>JAK2V617F</i> negative ET/PMF (Mut0) and B) <i>JAK2V617F</i>+ ET and PV; C) <i>JAK2V617F</i>+ ET and PMF; D) <i>JAK2V617F</i>+ PV and PMF. Table represents the genes exclusively expressed in a single MPN disorder.</p

Hierarchical clustering of highly significant genes expressed in CD34⁺ cells of MPNs.

<p>Statistical significance (p<0.01) was determined among <i>JAK2V617F</i> positive ET (yellow), PV (red), PMF (blue) and <i>JAK2V617F</i> negative ET/PMF (Mut0, green) by one way ANOVA. The numbers on top and below the color bar represent intensity of contrasts with offsets at 0 and 1. The total gene expression of MPNs is also clustered (above image) representing similarities among examined cells. The gene and array correlations were uncentered. The gene description is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135463#pone.0135463.t003" target="_blank">Table 3</a>.</p