Back ground: Aggressive NK-cell leukemia (ANKL) is a malignant NK-cell proliferative disorder resistant to most chemotherapies and frequently shows rapidly progressive clinical course. ANKL is characterized with hepatosplenomegaly, hemophagocytic syndrome, and strong association with EBV, and is mostly observed among Asian populations. Oncogenic mechanism of ANKL is not clear. NOTCH1 is a gene encoding a transmembrane receptor that regulates T-cell development and is a major oncogene in T-cell lymphoblastic leukemia (T-ALL). Recently, the potential involvement of NOTCH signaling in the development of NK-cells has been reported. We discuss here that the NOTCH1 protein might play a significant role in the pathogenesis of ANKL.

Patients, materials, and methods: 6 ANKL cases, 7 Chronic NK-cell lymphocytosis (CNKL) cases, and 3 NK tumor cell lines (NKL, NK-92, and KHYG) derived from ANKL patients were enrolled. Chromosomal translocation (7;9) was not found in any subjects. The expression levels of NOTCH1 intracellular subunit (NOTCH-IC) were examined with flowcytometry and western blotting. NK-cell lines were cultured with g-secretase inhibitor (GSI) (L-685,458) for evaluation of the effect. Cell proliferation and apoptosis were measured with XTT assay and annexinV positivity, respectively. The mutation analysis of NOTCH1 genome was performed with nested PCR and direct sequencing. mRNA expression of NOTCH1 gene was measured with real time PCR using the corresponding TaqMan probes.

Results: NOTCH-IC was detected in CD2+CD3− fractions from healthy donors, CNKL patients, ANKL patients, NK-cell lines, and Cos7 cells transfected with NOTCH1 gene coding NOTCH-IC, but not in CD2-CD3− fractions. The expression levels were significantly high in ANKL patients, compared to healthy donors (P < 0.05). With western blotting, cleaved form (110kDa) of NOTCH1 protein was recognized in all NK-cell lines. NOTCH-IC expression was reduced with the treatment of GSI in NKL and NK-92, but not in KHYG. Cell proliferation was inhibited with GSI in NKL and NK-92, whereas the inhibitory effect was not seen in KHYG. There was no significant difference in the increase ratio of apoptotic cells after the treatment of GSI among 3 NK-cell lines. Mutations in HD, TAD, or PEST domain, where frequent mutations are detected in T-ALL, were not found in NK-cell lines, and no enhanced expression of NOTCH1 mRNA in ANKL cells, compared with healthy donor NK-cells.

Conclusion: These results indicate that NOTCH-IC was increased in ANKL cells and might be an important role in the proliferation. Genomic or transcriptional dysregulation would not be the cause for enhanced expression of NOTCH1 in ANKL contrast with T-ALL. Further investigation is required for the relationship with NOTCH1 ligands. In the future, NOTCH1 might be a novel therapeutic target in ANKL.

Author notes

Disclosure: No relevant conflicts of interest to declare.