Baicalein generated two shRNAs that knocked down

Erentially required for the proliferation and survival of PMBL and HL lines with the 9p24 amplicon but are not essential genes in other DLBCL subtypes. Autocrine activation of JAK2 JAK2 protein was highly expressed in PMBL and HL lines with JAK2 amplification, and JAK2 phosphorylation was detected Baicalein exclusively in these cells. To test the requirement for JAK2 kinase activity, we treated lymphoma lines with a selective JAK2 inhibitor, TG101348. TG101348 reduced STAT6 phosphorylation in PMBL and HL lines, decreased viable cells in a dose dependent fashion and induced apoptosis in the same PMBL and HL lines that were sensitive to JAK2 knockdown. Like the JAK2 shRNA, TG101348 did not block cell cycle progression. Similar effects on cell viability and STAT6 signaling were obtained with another JAK2 inhibitor, AZD1480.
Antibody inhibition of IL 13 decreased STAT6 phosphorylation in all 5 HL lines and in all 3 PMBL lines. Interestingly, anti IL 13 also reduced the cell surface Clinofibrate expression of the IL 13 receptor chain in both cell types as did treatment with the JAK2 inhibitor TG101348, suggested that IL 13 secretion initiates a positive feedback loop that enhances IL 13 receptor expression and signaling in PMBL and HL cells. We generated two shRNAs that knocked down IL13R expression, reduced downstream signaling in PMBL cells, and were selectively toxic to PMBL and HL cells. We conclude that amplification and overexpression of JAK2 cooperates with autocrine IL 13 signaling to promote the survival of PMBL and HL cells. We next used both the JAK2 shRNA and TG101348 to identify genes regulated by JAK2 signaling in K1106 PMBL cells.
Remarkably, this JAK2 regulated gene signature accounted for roughly one sixth of the genes that were more highly expressed in primary PMBL tumors than in GCB DLBCL tumors, a highly significant overlap. Most of these genes were more highly expressed in PMBL cases with the 9p24 amplicon than in cases with wild type 9p24, indicating that this genetic abnormality has a broad influence on the signaling output of the JAK2 pathway. Of note, PMBL cases with wild type 9p24 copy number still had higher expression of these JAK2 regulated genes than did GCB DLBCLs, indicating that JAK2 signaling imparts a pervasive phenotype in a majority of PMBL tumors that is augmented by the 9p24 amplicon.
Moreover, a majority of these JAK2 regulated genes were also more highly expressed in HL lines than in GCB DLBCL lines, demonstrating that JAK2 signaling significantly shapes the biology of HL as well. Functional cooperation between JAK2 and JMJD2C Since both JAK2 and JMJD2C can modify the genome epigenetically, we focused our subsequent work on the mechanism by which these two amplicon genes might jointly alter PMBL and HL biology. Our interest in JAK2 and JMJD2C was further stimulated by a tissue microarray analysis that demonstrated high expression of these proteins in 70% and 38% of PMBL biopsies, respectively, but in significantly fewer biopsies of other DLBCL subtypes. We investigated whether JAK2 and JMJD2C might cooperatively sustain the survival and proliferation of PMBL and HL cells. To test this, we infected a population of cells with vectors expressing an shRNA targeting JMJD2C or a control shRNA together with GFP, and treated the cells

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