Aberrant Expression of IL-2R in Pediatric Anaplastic Large Cell Lymphoma: Cellular and Proteomic Analysis of Denileukin Diftitox (ONTAK) as a Potential Therapeutic Agent.

Constitutive expression of the NPM/ALK tyrosine kinase is a key oncogenic event in the pathogenesis of anaplastic large cell lymphoma (ALCL). Our previous cDNA microarray analysis of pediatric ALCLs obtained from the Cooperative Human Tissue Network (CHTN) demonstrated overexpression of IL-2Rγ in NPM/ALK-positive ALCLs relative to NPM/ALK-negative lymphomas and reactive lymphoid tissues. Furthermore, the mRNA levels of IL-2Rγ were overexpressed in Jurkat cells transfected with NPM/ALK relative to the vector-transfected cells.

To determine the feasibility of targeting IL-2R by ONTAK for treatment of pediatric ALCLs, we determined the prevalence of IL-2R expression in pediatric ALCLs and correlated its expression with that of the ALK protein. We used formalin-fixed paraffin-embedded tissues of ALCLs obtained from the CCG5941 clinical trial involving the pediatric population and determined the expression of ALK and CD25 (IL-2R) using immunohistochemistry. The effect of ONTAK on the viability of 5 cell lines derived from t(2;5)-positive ALCLs was assessed using an assay based on the cleavage of the tetrazolium salt WTS-1 by mitochondrial dehydrogenases. Western blot analysis was performed to determine the effect of ONTAK on cell cycle proteins and apoptosis. We utilized an endoproteinase-catalyzed ¹⁶O/¹⁸O differential isotopic strategy to quantitatively determine the global proteomic sequelae of IL-2R inhibition using ONTAK. Proteins were collected from the cell lysates of treated and non-treated SUDHL1 cells, subjected to ¹⁶O/¹⁸O labeling and then analyzed by reverse-phase liquid chromatography and electrospray ionization tandem mass spectrometry.

Analysis of 40 cases of ALCL revealed nuclear and cytoplasmic expression of ALK in the majority of cases. Strong membranous and cytoplasmic expression of CD25 was present in 27/40 (77%) cases while the reactive lymphocytes showed negligible or weak expression. Treatment of SUDHL-1 cells with ONTAK resulted in time and dose-dependent decrease in cell proliferation as measured by the WTS assay which was associated with induction of p27Kip1 and cleavage of PARP. A total of 253 proteins with 2 or more unique peptides were identified as being differentially expressed between treated and non-treated SUDHL-1 cells. Proteins of diverse location and function were identified. Importantly a large number of proteins known to be altered by diphtheria toxin and important in protein translation were underexpressed in treated cells (eIF2C, hnRNP, BRCA1, CREG, NuMA, 60S ribosomal protein L8). In addition novel proteins with transcriptional and cell signaling activities were identified, representing unique pathways that may be affected by IL-2 signaling.

Our studies demonstrate the constitutive expression of CD25 in the majority of pediatric ALK positive ALCLs. Our quantitative proteomic studies reveal novel insights into the signaling pathways involved in IL-2 signaling in ALCL cells. Our in vitro studies indicate that targeting of CD25 by ONTAK may represent a rational approach for treatment of pediatric patients with ALCL.