Functional validation of the anaplastic lymphoma kinase signature identifies CEBPB and Bcl2A1 as critical target genes
Roberto Piva, … , Antonino Neri, Giorgio Inghirami
Roberto Piva, … , Antonino Neri, Giorgio Inghirami
Published December 1, 2006
Citation Information: J Clin Invest. 2006;116(12):3171-3182. https://doi.org/10.1172/JCI29401.
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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 1

Inducible NPM-ALK knock down leads to apoptosis of ALCL cells and tumor regression in vivo.

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Inducible NPM-ALK knock down leads to apoptosis of ALCL cells and tumor ...
(A) Conditional expression of the shRNA internal marker gene GFP. TS cells cotransduced with pLV-tTR-KRAB/DsRed and pLVTH-ALK-A5/GFP (TS A5, right) or with the mutated pLVTH-ALK-A5M/GFP (TS A5M, left) lentiviral preparations were cultured with doxycycline (DOX) (1 μg/ml) for the indicated times and subsequently analyzed by flow cytometry (72 hours). (B) Kinetics of NPM-ALK knock down. TS-TTA-A5 cells were treated with DOX and harvested at the indicated times. Whole-cell lysates were analyzed by Western blotting with anti-GFP or ALK antibodies. (C and D) Inducible NPM-ALK knock down leads to downmodulation of ALK and of known downstream targets in TS-TTA-A5 cells. (C) RNA levels of NPM-ALK, CD30 (TNFRSF8), and β2-microglobulin transcripts were determined by semiquantitative RT-PCR (72 hours). (D) Protein expressions were assayed by Western blotting with the specified antibodies (72–84 hours). (E and F) Loss of NPM-ALK leads to G1 cell-cycle arrest and apoptosis. TS-TTA-A5 or -A5M cells were harvested after DOX induction and analyzed by flow cytometry. (E) The percentage of cells in G0/G1, S, and G2-M phases was determined by PI staining (96 hours). (F) The fraction of apoptotic cells was assayed by annexin V staining (120 hours). (G) NPM-ALK ablation induces tumor regression in vivo. Representative tumor growth curves for TS-TTA-A5 cells injected subcutaneously into BNX mice in an untreated mouse (green) or 2 mice treated for 14 days with DOX (red) starting at day 17. Each data point represents the average diameter of 2 tumor masses. Similar results were obtained from 9 mice injected with TS-TTA-A5 cells, with slightly differing kinetics.