Functional validation of the anaplastic lymphoma kinase signature identifies CEBPB and Bcl2A1 as critical target genes
Roberto Piva, Elisa Pellegrino, Michela Mattioli, Luca Agnelli, Luigia Lombardi, Francesco Boccalatte, Giulia Costa, Bruce A. Ruggeri, Mangeng Cheng, Roberto Chiarle, Giorgio Palestro, Antonino Neri, Giorgio Inghirami
Roberto Piva, Elisa Pellegrino, Michela Mattioli, Luca Agnelli, Luigia Lombardi, Francesco Boccalatte, Giulia Costa, Bruce A. Ruggeri, Mangeng Cheng, Roberto Chiarle, Giorgio Palestro, Antonino Neri, Giorgio Inghirami
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Research Article Oncology

Functional validation of the anaplastic lymphoma kinase signature identifies CEBPB and Bcl2A1 as critical target genes

Abstract

Anaplastic large cell lymphomas (ALCLs) represent a subset of lymphomas in which the anaplastic lymphoma kinase (ALK) gene is frequently fused to the nucleophosmin (NPM) gene. We previously demonstrated that the constitutive phosphorylation of ALK chimeric proteins is sufficient to induce cellular transformation in vitro and in vivo and that ALK activity is strictly required for the survival of ALK-positive ALCL cells. To elucidate the signaling pathways required for ALK-mediated transformation and tumor maintenance, we analyzed the transcriptomes of multiple ALK-positive ALCL cell lines, abrogating their ALK-mediated signaling by inducible ALK RNA interference (RNAi) or with potent and cell-permeable ALK inhibitors. Transcripts derived from the gene expression profiling (GEP) analysis uncovered a reproducible signature, which included a novel group of ALK-regulated genes. Functional RNAi screening on a set of these ALK transcriptional targets revealed that the transcription factor C/EBPβ and the antiapoptotic protein BCL2A1 are absolutely necessary to induce cell transformation and/or to sustain the growth and survival of ALK-positive ALCL cells. Thus, we proved that an experimentally controlled and functionally validated GEP analysis represents a powerful tool to identify novel pathogenetic networks and validate biologically suitable target genes for therapeutic interventions.

Authors

Roberto Piva, Elisa Pellegrino, Michela Mattioli, Luca Agnelli, Luigia Lombardi, Francesco Boccalatte, Giulia Costa, Bruce A. Ruggeri, Mangeng Cheng, Roberto Chiarle, Giorgio Palestro, Antonino Neri, Giorgio Inghirami

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Figure 5

CEBPB is an NPM-ALK and STAT3 target gene.

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CEBPB is an NPM-ALK and STAT3 target gene. (A) Identifi...
(A) Identification of genes whose expression strongly correlated with TNFRSF8 expression. Genes from the list obtained by the supervised analysis of TS-TTA-A5 (untreated+A1) versus A2+A3 cells (268 genes) were hierarchically clustered in all experimental conditions (33 samples). The plot represents a portion of the list, which includes transcripts with the highest degree of correlation to TNFRSF8 expression (green rectangle). (B) CEBPB expression is correlated with TNFRSF8. TNFRSF8 and CEBPB mRNA expression levels are represented in the histogram as assessed by microarray analysis of 12 TS-TTA-A5 samples. The statistical significance was ranked according to the z score. (C) C/EBPβ protein expression is strongly repressed following NPM-ALK silencing. TS-TTA and Su-DHL1-TTA cells were transduced with A5 or A5M and treated with DOX (84 hours). Western blot analysis was carried out on whole-cell lysates with specific antibodies, as indicated. Different bands represent multiple C/EBPβ isoforms. (D) STAT3 and ERK1/ERK2 regulate C/EBPβ expression. TS-TTA cells were transduced with pLVTH-STAT3, pLVTH-ERK1 and pLVTH-ERK2, pLVTH-AKT1, or pLVTH-PLC-γ shRNA interfering constructs and treated with DOX (96 hours). Western blot analysis was carried out on whole-cell lysates with the specified antibodies. (E) C/EBPβ protein expression is restricted to ALK-positive ALCL cell lines. C/EBPβ and NPM-ALK expression was determined by Western blot analysis on a panel of lymphoma/leukemia cell lines. α-Tubulin protein expression was included for protein normalization.