Oncogenic Kinase NPM/ALK Induces Immunosuppressive Protein CD274 (PD-LI, B7-H1) through STAT3 Expression
A Goradia, M Marzec, Q Zhang, P Raghunath, X Liu, M Paessler, H Wang, M Wysocka, M Wasik. Hospital of the University of Pennsylvania, Philadelphia, PA
Background: The mechanisms of malignant cell transformation caused by the oncogenic, chimeric NPM/ALK tyrosine kinase remain partially understood with most studies focusing on the impact of NPM/ALK on cell survival and proliferation. Here we show that the NPM/ALK+ T-cell lymphoma (ALK+ TCL) cells strongly express the immunosuppressive cell-surface protein CD274 (PD-L1, B7-H1) as determined on the mRNA and protein level.
Design: A multifaceted approach was employed using the ALK+TCL cell lines and tissues. NPM/ALK-dependent gene expression was determined using CEP-14083 ALK inhibitor and U133 Plus 2.0 array chips (Affymetrix) microarray analysis and confirmatory RT-PCR (on the RNA level) and flow cytometry and immunohistochemistry (on the protein level). The association of the NPM/ALK-activated STAT3 transcription factor with the PD-L1 gene promoter was determined using electromobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP).
Results: The CD274 expression is strictly dependent on the expression and enzymatic activity of NPM/ALK as demonstrated by inhibition of the NPM/ALK function in ALK+ TCL cells by the small molecule ALK inhibitor CEP-14083 and by documenting CD274 expression in IL-3-starved BaF3 cells transfected with the wild-type NPM/ALK but not the kinase-inactive NPM/ALK K210R mutant or empty vector alone. Immunohistochemical analysis with anti-CD274 antibodies demonstrated selective expression of PD-L1 in ALK+ TCL tissues.
NPM/ALK acts through activation of its key signal transmitter, transcription factor STAT3. STAT3 binds to the CD274 gene promoter in vitro and in vivo as shown in the EMSA and ChIP assays and is required for PD-L1 gene expression as demonstrated by siRNA-mediated STAT3 depletion.
Conclusions: These findings identify a novel mechanism of NPM/ALK-induced malignant cell transformation, based on the induction of CD274 expression and represent the first documented direct link between an oncoprotein and an immunosuppressive cell-surface protein.
Wednesday, March 11, 2009 9:30 AM
Poster Session V # 185, Wednesday Morning