Design and evaluation of chemically synthesized siRNA targeting the NPM-ALK fusion site in anaplastic large cell lymphoma (ALCL).
The NPM-ALK fusion protein is found in up to 75% of pediatric anaplastic large cell lymphomas (ALCL). The ALK kinase becomes constitutively activated and triggers malignant transformation. Molecular targeting of the tumor-specific NPM-ALK fusion by gene-silencing methods seems to be a promising approach both for the treatment of ALCL and to decipher signaling pathways used by NPM-ALK. We designed and evaluated three chemically synthesized small interfering RNAs (siRNAs) for downregulation of the NPM-ALK fusion mRNA. Compared to HeLa cells transfected with the NPM-ALK expression plasmid only and to an siRNA containing two point mutations, the most potent anti-NPM-ALK siRNA reduced NPM-ALK protein expression in HeLa cells to almost undetectable levels, and the number of cells stained positively for NPM-ALK decreased by 80%. With respect to signaling, expressing of NPM-ALK increased the activity of AKT and ERK in HeLa cells, and this effect could be blocked by the specific siRNA targeting NPM-ALK. Expression of endogenous NPM-ALK mRNA in SR786 ALCL cells decreased by 50%-60% in cells transfected with the NPM-ALK siRNA. However, the amount of NPM-ALK protein was not influenced by a single transfection of the siRNAs against NPM-ALK. Repeated transfections over 8 days led to a significant reduction in NPM-ALK protein but without induction of apoptosis. We believe that the long protein half-life of NPM-ALK, at least 48 hours, limits the application of transiently transfected siRNAs. Nevertheless, RNA interference (RNAi) offers a suitable technique to dissect signaling pathways employed by NPM-ALK and may potentially be used to develop siRNA-based gene therapeutic approaches against NPM-ALK-positive lymphomas.
Department of Pediatric Hematology and Oncology, Justus-Liebig-University, Giessen, Germany.
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Ritter
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Damm-Welk
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Fuchs
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RM
Bohle
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Borkhardt
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Woessmann
D001483:Base Sequence; D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D016403:Lymphoma, Large B-Cell, Diffuse / Q000235:genetics*; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D034741:RNA, Small Interfering / Q000138:chemical synthesis / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics; D014162:Transfection / Q000379:methods
D018076:DNA, Complementary; D015514:Oncogene Proteins, Fusion; D034741:RNA, Small Interfering; C107023:p80(NPM-ALK) protein; D011505:Protein-Tyrosine Kinases