Constitutive Activation of FRAP/mTOR Pathway in Pediatric Anaplastic Large Cell Lymphomas: Potential Role as a Therapeutic Target.

Constitutive expression of the chimeric NPM/ALK fusion protein encoded by the t(2;5)(p23;q35) is a key oncogenic event in the majority of pediatric anaplastic large cell lymphomas (ALCL). To determine the pathogenetic mechanisms involved in NPM/ALK-mediated lymphomagenesis we employed a mass spectrometry (MS)-based proteomics approach to identify changes in protein expression caused by the overexpression of NPM/ALK. We identified many proteins which were downstream targets of the FRAP/mTOR pathway including ribosomal S6 kinase (1.6-fold), translational initiation factor eIF (4.8-fold), ribosomal protein L11 (4.8-fold), eukaryotic translation initiation factor 3 (3.2-fold), translation initiation factor IF-2 homolog (4.3-fold) and translation initiation factor eIF-2alpha kinase (3.4-fold). The FRAP/mTOR pathway plays a key role in the regulation of cell growth and proliferation and positively regulates translation and ribosome biogenesis and is selectively inhibited by rapamycin.

To determine the feasibility of targeting the FRAP/mTOR pathway by rapamycin for treatment of pediatric ALCLs, we determined the prevalence of expression of key proteins in the FRAP/mTOR pathway in pediatric ALCLs and correlated its expression with that of the ALK protein. In addition we determined the in vitro effect of rapamycin on the viability of cell lines derived from t(2;5)-positive ALCLs.

We used formalin-fixed paraffin-embedded tissues of ALK-positive ALCLs (n=18) obtained from the Children’s Oncology Group clinical trials (CCG5941 and ANHL0131) and determined the expression of phospho-mTOR, phospho-70S6Kinase and phospho-S6 ribosomal protein using immunohistochemistry. The effect of rapamycin on the viability of cell lines derived from t(2;5)-positive ALCLs was determined by MTT assay and cell cycle analysis. Western blot analysis was performed to determine the effect of rapamycin on cell cycle proteins and apoptosis.

Immunohistochemical studies demonstrated diffuse strong nuclear expression of phospho-mTOR in 17/18 cases, and nuclear and cytoplasmic phospho-70S6kinase expression in 15/18 cases. In addition, cytoplasmic expression of phospho-S6 ribosomal protein was observed in 18/18 (100%) of cases. Importantly, the reactive lymphocytes demonstrated negligible expression of all three proteins. Furthermore, rapamycin potently decreased the viability of SUDHL-1 cells (30% reduction by 10nM at 48 hours) and resulted in G₁ cell cycle arrest without induction of caspase-3 activity. Western blot analysis demonstrated a reduction in the level of phospho-p70S6Kinase as well as 4EBP-1 levels.

Our studies demonstrate overexpression of many proteins in the FRAP/mTOR pathway in NPM/ALK-positive ALCLs. Our data indicate that the majority of pediatric ALCLs express proteins in the FRAP/mTOR pathway and are constitutively activated. Furthermore, our in vitro data support the use of rapamycin as a therapeutic agent in ALK-positive ALCLs.