NPM–ALK-Induced Reprogramming of Mature TCR-Stimulated T Cells Results in Dedifferentiation and Malignant Transformation

Abstract

Fusion genes including NPM–ALK can promote T-cell transformation, but the signals required to drive a healthy T cell to become malignant remain undefined. In this study, we introduce NPM–ALK into primary human T cells and demonstrate induction of the epithelial-to-mesenchymal transition (EMT) program, attenuation of most T-cell effector programs, reemergence of an immature epigenomic profile, and dynamic regulation of c-Myc, E2F, and PI3K/mTOR signaling pathways early during transformation. A mutant of NPM–ALK failed to bind several signaling complexes including GRB2/SOS, SHC1, SHC4, and UBASH3B and was unable to transform T cells. Finally, T-cell receptor (TCR)–generated signals were required to achieve T-cell transformation, explaining how healthy individuals can harbor T cells with NPM–ALK translocations. These findings describe the fundamental mechanisms of NPM–ALK-mediated oncogenesis and may serve as a model to better understand factors that regulate tumor formation. Significance: This investigation into malignant transformation of T cells uncovers a requirement for TCR triggering, elucidates integral signaling complexes nucleated by NPM–ALK, and delineates dynamic transcriptional changes as a T cell transforms. See related commentary by Spasevska and Myklebust, p. 3160

Regulation of CD45 phosphatase by oncogenic ALK in anaplastic large cell lymphoma

“…Pawlicki et al. have recently demonstrated that the reprogramming induced by NPM-ALK is central for T cell transformation and full acquisition of a neoplastic phenotype ( 26 ). However, in contrast to other TCR related molecules, such as CD3, LCK or ZAP70, CD45 is only downregulated and not completely repressed because altered levels of CD45 can affect T cell development within the thymus and T cell survival in the periphery ( 32 – 34 ).…”

“…Specifically, the CD45RO isoform was affected by NPM-ALK, whereas the CD45RA isoform was not, as observed by flow cytometry and western blot analyses (Figure 2B and Supplementary Figures 1A, B). We and others previously reported that ALK oncogenic activity controls protein expression through transcriptional regulation or epigenetic silencing (6,25,26). We then investigated the mechanisms of ALK regulation on CD45 expression in ALCL cell lines.…”

Combined disruption of T cell inflammatory regulators Regnase-1 and Roquin-1 enhances antitumor activity of engineered human T cells

“…A total of 2 × 10 6 T cells were cultured in 2 mL R10 media in 12-well plates and replated in fresh R10 media every 3 to 4 d for 28 d. At each medium change, cells were maintained at a concentration of 1 × 10 6 cells/mL and counted using the Countess 3 FL Automated Cell Counter (Invitrogen) using Trypan blue (Gibco) as a viability stain. NPM–ALK-transduced T cells were used as a positive control for transformed T cells and have been described previously ( 34 , 35 ).…”

De novo generation of the NPM-ALK fusion recapitulates the pleiotropic phenotypes of ALK+ ALCL pathogenesis and reveals the ROR2 receptor as target for tumor cells

“…The majority of ALK+ ALCL tumor cells have been shown to express at least one T-cell specific marker. This pleiotropic combination of markers observed in ALK+ ALCL has rendered the identification of the cell of origin difficult, with reports proposing either a thymic or a peripheral origin [ 6 – 8 ].…”

“…Several in vivo approaches in mice have failed to phenocopy human ALK+ ALCL and rather often yield B-cell lymphomas [ 12 – 16 ]. More recently, transduction of primary human CD4+ T lymphocytes with the NPM-ALK transgene has led to in vitro transformation of the cells [ 7 , 8 , 17 ] and in vivo tumor formation [ 7 , 17 ]. However, lentiviral expression does not precisely reproduce gene dosage nor the spatiotemporal variations in gene expression associated with the various stages of differentiation.…”