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 4

BCL2A1 expression is regulated by NPM-ALK activity and sustains the survival of ALK-positive ALCL cells.

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BCL2A1 expression is regulated by NPM-ALK activity and sustains the surv...
(A and B) BCL2A1 shRNA induces apoptosis of ALCL cells. (A) TS cells were transduced with lentivirus (pLKO-GFP) expressing 4 BCL2A1-directed shRNAs (2A–D). BCL2A1 mRNA expression was determined by RT-PCR analysis (72 hours). (B) TS cells were transduced with a mock or a BCL2A1-directed shRNA (2C), and the percentage of apoptotic cells was determined by tetrametylrodamine methyl ester (TMRM) staining 5 and 7 days after infection. Cell morphology of BCL2A1- and mock shRNA–transduced TS cells is shown in the inset. These findings are representative of 3 independent experiments. (C–E) BCL2A1 expression correlates with ALK signaling. (C) The expression of BCL2 family genes was evaluated in all experimental conditions (33 samples) for absolute correlation with ALK. The BCL2A1 gene clustered within the branch including the ALK probe set (green rectangle). (D) BCL2A1 mRNA expression is significantly downmodulated after ALK RNAi. mRNA expression levels were assessed by microarray analysis of ALK-knockdown experiments in TS and Su-DHL1 cells. (E) Loss of ALK expression leads to the BCL2A1 protein downregulation. TS-TTA and Su-DHL1-TTA cells transduced with A5 or A5M were treated with DOX (84 hours). Western blot analysis was carried out on whole-cell lysates with antibodies against BCL2A1 and ALK.